Modulation of axon degeneration

ABSTRACT

The invention relates generally to treatment of neurological disorders and nervous system injuries. The invention specifically provides methods of using modulators of particular target proteins to modulate degeneration of neurons or portions thereof, such as axons.

FIELD OF THE INVENTION

This invention relates generally to treatment of neurological disordersand nervous system injuries. The invention specifically concerns the useof modulators of particular target proteins and processes in methods toinhibit neuron and axon degeneration.

BACKGROUND OF THE INVENTION

Neuron or axon degeneration plays a central role in the properdevelopment of the nervous system and is a hallmark of manyneurodegenerative diseases including, for example, amyotrophic lateralsclerosis (ALS), Alzheimer's disease, and Parkinson's disease, as wellas traumatic injury to the brain and spinal cord. These diseases andinjuries are devastating to patients and caregivers, and also result ingreat financial burdens, with annual costs currently exceeding severalhundred billion dollars in the United States alone. Most currenttreatments for these diseases and conditions are inadequate. Adding tothe urgency of the problems created by these diseases is the fact thatmany such diseases are age-related, and thus their incidence isincreasing rapidly as population demographics change. There is a greatneed for the development of effective approaches to treatingneurodegenerative diseases and nervous system injuries.

SUMMARY OF THE INVENTION

The invention provides methods for inhibiting degeneration of a neuronor a portion thereof (e.g., the neuron cell body, an axon, or adendrite). The methods involve administering to the neuron or portionthereof an agent that modulates: (i) the activity or expression of atarget protein in the neuron or portion thereof, or (ii) a process inthe neuron or portion thereof.

Examples of proteins that can be targeted in the methods of theinvention include dual leucine zipper-bearing kinase (DLK), glycogensynthase kinase 3β (GSK3β), p38 mitogen-activated protein kinase (p38MAPK), β-catenin, transcription factor 4 (TCF4), epidermal growth factorreceptor (EGFR), phosphoinositide 3-kinase (PI3K), cyclin-dependentkinase 5 (cdk5), adenylyl cyclase, c-Jun N-terminal kinase (JNK),BCL2-associated X protein (Bax), Ih channel,calcium/calmodulin-dependent protein kinase kinase (CaMKK), G-proteins,G-protein coupled receptors, transcription factor 4 (TCF4), orβ-catenin, while examples of processes that can be targeted aretranscription and protein synthesis.

The neuron or portion thereof can consist of or can be within a neuronselected from the group consisting of a cerebellar granule neuron, adorsal root ganglion neuron, a cortical neuron, a sympathetic neuron,and a hippocampal neuron.

The agents can be, for example, inhibitors of the target protein orprocess. Further, the agents can be, for example, selected from thegroup consisting of antibodies, polypeptides, peptides, peptibodies,nucleic acid molecules, short interfering RNAs (siRNAs),polynucleotides, aptamers, small molecules, and polysaccharides. In thecase of antibodies, the antibodies can be monoclonal antibodies,chimeric antibodies, humanized antibodies, human antibodies, or antibodyfragments (e.g., an Fv, Fab, Fab′, or F(ab′)₂ fragment). In the case ofsmall molecules, the agent can be, for example, selected from the groupconsisting of MG132, SB 415286, GSK3β inhibitor I, GSK33 inhibitor VII,GSK3β inhibitor VIII, GSK3β inhibitor XII, Lithium Chloride, SB 202190,SB 239063, SB 239069, SB 203580, SB 203580 HCl, AG 556, AG 555, AG 494,PD168393, Tyrphostin B44, Tyrphostin B42 (AG 490), LY 294022,Anisomycin, Cycloheximide, Roscovitine, Forskolin, NKH 477, ActinomycinD, SP600125, Bax Channel Blocker, ZD7288, STO-609, bortezomid,disulfiram, pamapimod, gefitinib, erlotinib, lapatinib ditosylate,demeclocycline hydrochloride, gentamicin sulfate, neomycin sulfate,paromomycin sulfate, and pharmaceutically acceptable salts thereof.

The neuron or portion thereof can be present in a subject, such as ahuman subject. The subject can, for example, have or be at risk ofdeveloping a disease or condition selected from the group consisting of(i) disorders of the nervous system, (ii) conditions of the nervoussystem that are secondary to a disease, condition, or therapy having aprimary effect outside of the nervous system, (iii) injuries to thenervous system caused by physical, mechanical, or chemical trauma, (iv)pain, (v) ocular-related neurodegeneration, (vi) memory loss, and (vii)psychiatric disorders.

Examples of disorders of the nervous system include amyotrophic lateralsclerosis (ALS), trigeminal neuralgia, glossopharyngeal neuralgia,Bell's Palsy, myasthenia gravis, muscular dystrophy, progressivemuscular atrophy, primary lateral sclerosis (PLS), pseudobulbar palsy,progressive bulbar palsy, spinal muscular atrophy, inherited muscularatrophy, invertebrate disk syndromes, cervical spondylosis, plexusdisorders, thoracic outlet destruction syndromes, peripheralneuropathies, prophyria, Alzheimer's disease, Huntington's disease,Parkinson's disease, Parkinson's-plus diseases, multiple system atrophy,progressive supranuclear palsy, corticobasal degeneration, dementia withLewy bodies, frontotemporal dementia, demyelinating diseases,Guillain-Barré syndrome, multiple sclerosis, Charcot-Marie-Toothdisease, prion disease, Creutzfeldt-Jakob disease,Gerstmann-Sträussler-Scheinker syndrome (GSS), fatal familial insomnia(FFI), bovine spongiform encephalopathy, Pick's disease, epilepsy, andAIDS demential complex.

Examples of pain include chronic pain, fibromyalgia, spinal pain, carpeltunnel syndrome, pain from cancer, arthritis, sciatica, headaches, painfrom surgery, muscle spasms, back pain, visceral pain, pain from injury,dental pain, neuralgia, such as neuogenic or neuropathic pain, nerveinflammation or damage, shingles, herniated disc, torn ligament, anddiabetes.

Examples of conditions of the nervous system that are secondary to adisease, condition, or therapy having a primary effect outside of thenervous system include peripheral neuropathy or neuralgia caused bydiabetes, cancer, AIDS, hepatitis, kidney dysfunction, Colorado tickfever, diphtheria, HIV infection, leprosy, lyme disease, polyarteritisnodosa, rheumatoid arthritis, sarcoidosis, Sjogren syndrome, syphilis,systemic lupus erythematosus, and amyloidosis.

Examples of injuries to the nervous system caused by physical,mechanical, or chemical trauma include nerve damage caused by exposureto toxic compounds, heavy metals, industrial solvents, drugs,chemotherapeutic agents, dapsone, HIV medications, cholesterol loweringdrugs, heart or blood pressure medications, and metronidazole.Additional examples include burn, wound, surgery, accidents, ischemia,prolonged exposure to cold temperature, stroke, intracranial hemorrhage,and cerebral hemorrhage.

Examples of psychiatric disorders include schizophrenia, delusionaldisorder, schizoaffective disorder, schizopheniform, shared psychoticdisorder, psychosis, paranoid personality disorder, schizoid personalitydisorder, borderline personality disorder, anti-social personalitydisorder, narcissistic personality disorder, obsessive-compulsivedisorder, delirium, dementia, mood disorders, bipolar disorder,depression, stress disorder, panic disorder, agoraphobia, social phobia,post-traumatic stress disorder, anxiety disorder, and impulse controldisorders.

Examples of ocular-related neurodegeneration include glaucoma, latticedystrophy, retinitis pigmentosa, age-related macular degeneration (AMD),photoreceptor degeneration associated with wet or dry AMD, other retinaldegeneration, optic nerve drusen, optic neuropathy, and optic neuritis.Examples of glaucoma include primary glaucoma, low-tension glaucoma,primary angle-closure glaucoma, acute angle-closure glaucoma, chronicangle-closure glaucoma, intermittent angle-closure glaucoma, chronicopen-angle closure glaucoma, pigmentary glaucoma, exfoliation glaucoma,developmental glaucoma, secondary glaucoma, phacogenic glaucoma,glaucoma secondary to intraocular hemorrhage, traumatic glaucoma,neovascular glaucoma, drug-induced glaucoma, toxic glaucoma, andglaucoma associated with intraocular tumors, retinal deatchments, severechemical burns of the eye, and iris atrophy.

Contacting of the neuron or portion thereof with the agent, according tothe methods of the invention, can involve administering to a subject apharmaceutical composition including the agent. The administering can becarried out by, for example, intravenous infusion; injection byintravenous, intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial or intralesional routes; or topical or ocular application.Further, the methods of the invention can include administering to asubject one or more additional pharmaceutical agents.

In other examples, the neuron or portion thereof treated according tothe methods of the invention is ex vivo or in vitro (e.g., a nerve graftor nerve transplant). The invention also includes methods of identifyingagents for use in inhibiting degeneration of a neuron or a portionthereof. These methods involve contacting a neuron or portion thereofwith a candidate agent in an assay of axon or neuron degeneration (e.g.,anti-nerve growth factor (NGF) antibodies, serum deprivation/KClreduction, and/or rotenone treatment). Detection of reduced degenerationof the neuron or portion thereof in the presence of the candidate agent,relative to a control, indicates the identification of an agent for usein inhibiting degeneration of a neuron or portion thereof. The candidateagent can be, for example, selected from the group consisting ofantibodies, polypeptides, peptides, peptibodies, nucleic acid molecules,short interfering RNAs (siRNAs), polynucleotides, aptamers, smallmolecules, and polysaccharides.

The invention also includes pharmaceutical compositions and kits thatcontain one or more agent that can be used to inhibit degeneration of aneuron or a portion thereof, as described herein. The pharmaceuticalcompositions and kits can include, for example, one or more agentselected from the group consisting of MG132, SB 415286, GSK3β inhibitorI, GSK3β inhibitor VII, GSK3β inhibitor VIII, GSK3β inhibitor XII,Lithium Chloride, SB 202190, SB 239063, SB 239069, SB 203580, SB 203580HCl, AG 556, AG 555, AG 494, PD168393, Tyrphostin B44, Tyrphostin B42(AG 490), LY 294022, Anisomycin, Cycloheximide, Roscovitine, Forskolin,NKH 477, Actinomycin D, SP600125, Bax Channel Blocker, ZD7288, STO-609,bortezomid, disulfiram, pamapimod, gefitinib, erlotinib, lapatinibditosylate, demeclocycline hydrochloride, gentamicin sulfate, neomycinsulfate, paromomycin sulfate, and pharmaceutically acceptable saltsthereof. The pharmaceutical compositions and kits can optionally includeone or more pharmaceutically acceptable excipients. Further, thepharmaceutical compositions and kits can optionally include instructionsfor use of the compositions and kits in methods for inhibitingdegeneration of a neuron or portion thereof.

In any of the methods, compositions, and kits of the invention, theagent may be a DLK signaling inhibitor (e.g., a siRNA molecule targetingDLK comprising, e.g., the sequence of, desirably, GCACTGAATTGGACAACTCTT(SEQ ID NO: 1), GAGTTGTCCAATTCAGTGCTT (SEQ ID NO: 2),GGACATCGCCTCCGCTGATTT (SEQ ID NO: 3), or ATCAGCGGAGGCGATGTCCTT (SEQ IDNO: 4), or GCAAGACCCGTCACCGAAATT (SEQ ID NO: 5), TTTCGGTGACGGGTCTTGCTT(SEQ ID NO: 6), GCGGTGTCCTGGTCTACTATT (SEQ ID NO: 7), orTAGTAGACCAGGACACCGCTT (SEQ ID NO: 8); an antibody, such as, antibody317, antibody 318, antibody 319, antibody 320, antibody 321, antibody322, an siRNA molecule targeting the JNK1 sequence ofTTGGATGAAGCCATTAGACTA (SEQ ID NO: 9), an siRNA molecule targeting theJNK2 sequence of ACCTTTAATGGACAACATTAA (SEQ ID NO: 10) orAAGGATTAGCTTTGTATCATA (SEQ ID NO: 11), an siRNA targeting the JNK3sequence of CCCGCATGTGTCTGTATTCAA (SEQ ID NO: 12), SP600125, JNKVinhibitor, JNKVIII inhibitor, SC-202673, SY-CC-401, SP600125, As601245,XG-102, myricetin, T278A DLK, S281A DLK, S152A DLK, and the leucinezipper domain of DLK), a GSK3β inhibitor (e.g., SB415287, GSK3βinhibitor I, GSK3β inhibitor VII, GSK3β inhibitor VIII, GSK3β inhibitorXII, and lithium chloride), an EGFR pathway inhibitor (e.g., erlotinib,tyrphostin B44, tyrphostin B42/AG490, AG555, AG494, PD168393, SB203580,SB239063, SB202190, SB239069, STO-609, and SP600125), or a G-proteininhibitor (e.g., SCG292676 and pertussis toxin).

In any of the above-described methods, the administering of an agentresults in at least a 10% decrease (e.g., at least 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even100% decrease) in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8. 9, or 10)symptoms of a disorder of the nervous system; condition of the nervoussystem that is secondary to a disease, condition, or therapy having aprimary effect outside of the nervous system; injury to the nervoussystem caused by physical, mechanical, or chemical trauma; pain;ocular-related neurodegeneration; memory loss; or psychiatric disorder.Non-limiting examples of such symptoms include tremors, slowness ofmovement, ataxia, loss of balance, depression, decreased cognitivefunction, short-term memory loss, long-term memory loss, confusion,changes in personality, language difficulties, loss of sensoryperception, sensitivity to touch, numbness in extremities, muscleweakness, muscle paralysis, muscle cramps, muscle spasms, significantchanges in eating habits, excessive fear or worry, insomnia, delusions,hallucinations, fatigue, back pain, chest pain, digestive problems,headache, rapid heart rate, dizziness, blurred vision, shadows ormissing areas of vision, metamorphopsia, impairment in color vision,decreased recovery of visual function after exposure to bright light,and loss in visual contrast sensitivity.

In any of the above-described methods, the administering results in atleast a 10% decrease (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% decrease)in the likelihood of developing a disorder of the nervous system;condition of the nervous system that is secondary to a disease,condition, or therapy having a primary effect outside of the nervoussystem; injury to the nervous system caused by physical, mechanical, orchemical trauma; pain; ocular-related neurodegeneration; memory loss; orpsychiatric disorder, compared to a control population of subjects thatare not administered said agent.

The invention also provides methods for activating degeneration of aneuron or portion thereof. These methods involve administering to aneuron or portion thereof an agent that modulates: (i) the activity of atarget protein in the neuron or portion thereof, or (ii) a process inthe neuron or portion thereof. The target protein can be, for example,selected from the group consisting of dual leucine zipper-bearing kinase(DLK), glycogen synthase kinase 3β (GSK3β), p38 mitogen-activatedprotein kinase (p38 MAPK), epidermal growth factor receptor (EGFR),phosphoinositide 3-kinase (PI3K), cyclin-dependent kinase 5 (cdk5),adenylyl cyclase, c-Jun N-terminal kinase (JNK), BCL2-associated Xprotein (Bax), Ih channel, calcium/calmodulin-dependent protein kinasekinase (CaMKK), a G-protein, a G-protein coupled receptor, transcriptionfactor 4 (TCF4), or β-catenin, while the process can be, for example,transcription or protein synthesis. Further, the agent can be, forexample, an activator of the target protein or process (as is the casefor the targets listed above, with the exception of adenylyl cyclase).In the methods of activating degeneration of a neuron or portion thereofdescribed above, the modulation of a target protein may be an increasein the activity or expression of GSK3β, a decrease in the activity orexpression of β-catenin, and/or a loss in the activity or expression ofTCF4.

The invention also provides purified antibodies that specifically bindto the phosphorylated form of DLK (e.g., antibody 318, antibody 319,antibody 320, antibody 321, or antibody 322) and inhibitory nucleicacids (e.g., siRNA) that comprise the sequence of, desirably,GCACTGAATTGGACAACTCTT (SEQ ID NO: 1), GAGTTGTCCAATTCAGTGCTT (SEQ ID NO:2), GGACATCGCCTC CGCTGATTT (SEQ ID NO: 3), or ATCAGCGGAGGCGATGTCCTT (SEQID NO: 4), or GCAAGACCCGTCACCGAAATT (SEQ ID NO: 5), TTTCGGTGACGGGTCTTGCTT (SEQ ID NO: 6), GCGGTGTCCTGGTCTACTATT (SEQ ID NO: 7), orTAGTAGACCAGGACACCGCTT (SEQ ID NO: 8) that mediate a decrease in theexpression of DLK.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that treatment of neurons for 20 hours with anti-NGFantibodies results in axon degeneration. The two images in the top rowshow neurons visualized with Tuj1 (neuron specific β-tubulin)antibodies, with and without 20 hours of treatment with anti-NGFantibodies. The two images in the bottom row show neurons visualizedwith actin antibodies incubated with or without (control) 50 μg/mlanti-NGF antibodies.

FIG. 2 shows that varicosities form in neurons cultured with anti-NGFantibodies for 1, 3, 6, 9, 12, or 16 hours.

FIG. 3 shows that axons cultured with anti-NGF antibodies for 16 hourslack elongated mitochondria and show accumulation of mitochondria invaricosities.

FIG. 4 shows that, in axons cultured with anti-NGF antibodies for 0 to48 hours, the microtubule network is not disassembled before the actinor neurofilament networks.

FIG. 5 illustrates Wallerian degeneration, which takes place in axonssevered from neuron cell bodies (top panel; Raff et al., Science296(5569):868-871, 2002), and shows that there is a significant delay inaxon degeneration after lesion in Wallerian Degeneration Slow (WldS)mutants, as compared to controls (bottom panel; Araki et al., Science305(5686):1010-1013, 2004).

FIGS. 6A-6D show that a proteasome inhibitor and a GSK inhibitor preventaxon degeneration in an anti-NGF antibody-based NGF withdrawal assay.

FIGS. 7A-7D show that a p38 MAPK inhibitor and an adenylyl cyclaseactivator prevent axon degeneration in an anti-NGF antibody-based NGFwithdrawal assay.

FIGS. 8A-8D show that a transcription inhibitor and an EGFR kinaseinhibitor prevent axon degeneration in an anti-NGF antibody-based NGFwithdrawal assay.

FIGS. 9A-9D show that a JNK inhibitor and Bax Channel Blocker preventaxon degeneration in an anti-NGF antibody-based NGF withdrawal assay.

FIGS. 10A-10D show that an Ih channel blocker and a CAMKK inhibitorprevent axon degeneration in an anti-NGF antibody-based NGF withdrawalassay.

FIG. 11 is a graph showing the activities of inhibitors of GSK3 (30 μMSB415286), EGFR kinase (10 μM AG555), p38 MAPK (30 μM SB239063), CAMKK(15 μM STO-609), and JNK (10 μM SP600125) when added at the time of NGFwithdrawal (t=0) or at 3, 6, 9, or 12 hours after NGF withdrawal.

FIG. 12 illustrates a Campenot chamber, in which somal (cell body) andaxonal environments are separated.

FIG. 13 shows that axon degeneration is localized and proceeds withoutapoptosis in Campenot chamber studies in which NGF withdrawal took placein the axon-containing chamber. Degeneration is visualized by tubulinimmunofluorescence.

FIG. 14 shows that there are no signs of degeneration in the cell bodycompartment in the Campenot chamber-based assay illustrated in FIG. 13.

FIG. 15 shows that cell bodies (left panels), but not axons (rightpanels), in the presence of 30 μM SB415 (GSK inhibitor; GSKi) or 15 μMAct D (transcription inhibitor; TXNi) were protected from localdegeneration.

FIG. 16 shows that axons (right panels), but not cell bodies (leftpanels), exposed to anti-NGF antibodies in the presence of 10 μM AG555(EGFR inhibitor; EGFRi) or 30 μM SB239 (p38 inhibitor; p38i) wereprotected from local degeneration.

FIG. 17 is a graph showing quantification of axon degeneration inCampenot chambers in which anti-NGF antibodies were added to the axonalenvironment in the presence or absence (DMSO) of 15 μM actinomycin D(ActD), 30 μM SB415286 (SB415), 10 μM AG555, or 30 μM SB239063 (SB239)in the axonal (Axon) or the somal environment (Cell).

FIG. 18 is a model based on data from the screens described herein.

FIG. 19 shows that cell bodies appear smaller when NGF is removed fromthe axon compartment in a Campenot chamber.

FIG. 20 shows that many neurons deprived of NGF in the axon compartmenthave increased cleavage of caspase-3 and show nuclear condensation.(Neuron health may be affected by the membrane stain DiI.)

FIG. 21 is a graph showing GSK3 activity (as measured by decreasedlevels of phosphorylated GSK3β) at the start of NGF withdrawal (t=0) andafter 1, 3, 6, 9, and 12 hours of NGF withdrawal in the cell body oraxon compartment.

FIG. 22 is a graph showing JNK activity (as measured by increased levelsof phosphorylated JNK) at the start of NGF withdrawal (t=0) and after 1,3, 6, 9, and 12 hours of NGF withdrawal in the cell body or axoncompartment.

FIG. 23 shows that a large number of axons with varicosities, as well asfragmented axons, were observed when the cell body inhibitors (30 μMSB415 (GSKi) and 15 μM Act D (TXNi)) were added to the cell bodycompartment. The addition of the axon inhibitors (10 μM AG555 (EGFRi)and 30 μM SB239 (p38i)) to the axon compartment showed fewervaricosities, and the axons seemed to go straight to fragmentation.

FIG. 24 shows that there may be more functional mitochondria, but stillno elongated mitochondria, in NGF-deprived neurons treated with GSK,EGFR, and p38 inhibitors.

FIG. 25 shows that the GSK inhibitor SB415 can delay axon degenerationafter lesion.

FIG. 26 shows that, after global NGF withdrawal, 10 μM or 25 μM GSKinhibitor blocks axon degeneration, but does not block cell death.

FIG. 27 shows that EGFR expression is increased in a section of SOD1mouse (Tg) spinal cord stained with anti-EGFR antibodies (right panel),as compared to a non-transgenic control (NTG; left panel).

FIG. 28 shows that EGFR is normally expressed in neurons (motor neurons)and that the level of phosphorylated EGFR (pEGFR) is increased in ALSSOD1 mouse model (SOD1-Tg) compared to a non-transgenic control(Non-Tg).

FIG. 29 shows that the number of axons is decreased in the ALS SOD1mouse model (SOD1-Tg) as compared to a non-transgenic control (Non-Tg),and that the phosphorylated EGFR (pEGFR) in the ALS SOD1 model partiallyco-localizes with axons.

FIG. 30 shows that the small molecule inhibitors of JNK (5 μM SP600125),CaMKK (5 μM STO-609), EGFR (1 μM or 10 μM AG555), p38 (5 μM SB239063),and GSK (10 μM SB415286) protect cerebellar granule neurons from serumdeprivation/KCl reduction.

FIG. 31 shows that the small molecule inhibitors of EGFR, GSK, CaMKK,JNK, and p38 protect hippocampal neurons against 10 μM rotenone.

FIG. 32 shows that the small molecule inhibitors of EGFR, GSK, CaMKK,JNK, and p38 protect cortical neurons against 10 μM rotenone.

FIG. 33 shows that ErbB receptorsare detected on axons in dorsal rootganglion neurons by immunocytochemistry using antibodies specific forEGFR (top left panel), Her2 (top right panel), Her3 (bottom left panel),and Her4 (bottom right panel).

FIG. 34 shows that EGFR is expressed in axons of dorsal root ganglionneurons using immunocytochemistry.

FIG. 35 shows that 100 μg/mL EGF does not induce axon degeneration whenadded to dorsal root ganglion neurons and that addition of 100 μg/mL EGFinduces phosphorylation of ERK in the treated neurons.

FIG. 36 shows that the EGFR ectodomain (50 μg/mL) does not block axondegeneration induced by NGF withdrawal in dorsal root ganglion neurons.

FIG. 37 shows that 3.4 μM, 11.1 μM, 33.3 μM, and 100 μM Tarceva®(erlotinib) blocks degeneration in dorsal spinal cord explants.

FIG. 38 shows that dual leucine zipper-bearing kinase (DLK) actsupstream from JNK in axon degeneration. Transfection of a plasmidencoding wild type DLK in 293 cells results in JNK activation (asmeasured by increased levels of phosphorylated JNK) compared to cellsmock-transfected with a control plasmid or a plasmid encodingkinase-dead DLK (DLK-KD). Knockdown of DLK expression by siRNA in dorsalroot ganglion neurons protects axons from degeneration induced by NGFwithdrawal. The knockdown of DLK expression using DLK siRNA wasconfirmed using quantitative PCR as compared to a control siRNA (bottomright panel).

FIG. 39 shows that siRNA knockdown of DLK signaling delays local axondegeneration.

FIGS. 40A and 40B depict the results of experiments assessing the impactof DLK knockdown on NGF withdrawal-induced sympathetic neurondegeneration using phase contrast microscopy to visualize neurons.

FIG. 41 shows that knockdown of DLK expression using DLK siRNA (DLK)protects sympathetic neurons from camptothecin- and vincristine-inducedapoptosis compared to neurons treated with a control siRNA (NT).

FIG. 42 shows that transfection of sympathetic neurons with a plasmidencoding kinase-dead DLK (KD) protects the neurons from NGFwithdrawal-induced apoptosis compared to neurons transfected with aplasmid encoding wild type DLK (DLK).

FIGS. 43A-43D show the results of experiments assessing the bindingspecificities of the anti-pDLK antibodies described in Example 15A. FIG.43A shows Western blot analyses of the binding of each of the anti-pDLKantibodies described herein to DLK, DLK in the presence of a dominantnegative DLK, and control kinase MLK3. FIG. 43B shows immunofluorescentmicroscopic images of the binding of anti-pDLK antibodies 318 and 319 tocultured 293T cells transformed with DLK (upper two images) or controlkinase MLK3 (lower two images). FIGS. 43C and 43D show Western blotsusing JNK and phospho-JNK antibodies.

FIGS. 44A and 44B show binding of anti-pDLK antibodies (antibody 318) tospinal cord sections in wild type and SOD1 mutant mice at end stage ofdisease (FIG. 43A) and at the onset of symptoms (FIG. 44B).

FIG. 44C depicts Western blot analyses of pDLK, pJNK, and pcJUN levelsin human Alzheimer's disease patient cortical samples.

FIGS. 45A and 45B depict the results of experiments assessing the impactof DLK silencing on phosphorylation of JNK in response to NGF withdrawalstress in sympathetic neurons and dorsal root ganglion neurons; andvincristine-induced stress in cortical neurons, as described in Example15C and Example 14B.

FIG. 46 shows the protective effect of JNK inhibitors on DRG explantssubjected to NGF withdrawal stress, as described in Example 15C.

FIG. 47 depicts the results of experiments assessing the impact ofsilencing JNK1, JNK2, JNK3, individually, and JNK2 and JNK3 together inDRG neurons on axon degeneration observed upon NGF withdrawal stress, asdescribed in Example 15C.

FIG. 48A shows the affect of DLK siRNA and control siRNA on the survivalof cortical neurons.

FIG. 48B shows the affect of DLK siRNA and control siRNA on the survivalof sympathetic neurons.

FIG. 49 is a set of immunomicrographs showing the ability of aninhibitor of G-coupled protein receptors (SCH 202676; 10 μM or 100 μM)to prevent NGF withdrawal-induced degeneration in DRGs.

FIG. 50 is a set of immunomicrographs showing the ability of SCH 202676(0.1 μM or 1 μM) to prevent NGF withdrawal-induced degeneration in DRGs.

FIG. 51 is a set of immunomicrographs showing the ability of 0.01 μg/mL,0.1 μg/mL, or 1 μg/mL pertussis toxin (an inhibitor of G-proteinsignaling) to prevent NGF withdrawal-induced degeneration in DRGs.

FIG. 52 is a set of immunomicrographs of rat hippocampal neurons showingthe effect of expression of active mutant GSK (GSK3S9A), wild type TCF4,and mutant inactive TCF4 on degeneration.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

The term “target” is used herein to refer to proteins and processesthat, when modulated by agents impacting their activities, inhibit ordecrease axon degeneration. Most of the targets described herein, whencontacted with an agent that inhibits their activity, inhibit ordecrease axon degeneration, but the targets of the present inventionalso include proteins and processes that, when activated, inhibit ordecrease axon degeneration. Exemplary targets of the invention are asfollows: dual leucine zipper-bearing kinase (DLK), glycogen synthasekinase 3β (GSK3β), p38 mitogen-activated protein kinase (p38 MAPK),epidermal growth factor receptor (EGFR), phosphoinositide 3 kinase(PI3K), cyclin-dependent kinase 5 (Cdk5), adenylyl cyclase, c-JunN-terminal kinase (JNK), BCL2-associated X protein (Bax), Ih channel,calcium/calmodulin-dependent protein kinase kinase (CaMKK), a G-protein,a G-protein coupled receptor, transcription factor 4 (TCF4), β-catenin,transcription, and protein synthesis. A selection of common alternatedesignations for several of these targets is listed in Table 1. Thetargets include native, human sequences and homologues of thesesequences from monkeys, mice, rats, and other non-human mammals,including all naturally occurring variants, such as alternativelyspliced and allelic variants and isoforms, as well as soluble formsthereof. Exemplary, non-limiting sequence references are also providedin Table 1. Additional sequences, including sequences of various targetisoforms, variants, homologues, and fragments may also be considered astargets, according to the present invention.

TABLE 1 Exemplary Genbank Target Alternate names Accession numbersGycogen synthase GSK-3β; GSK-3 beta 3; GSK3beta isoform; CAG38748 kinase3 beta (GSK3β) GSK-3α, GSK-3b2 NP_002084 NP_063937 β-catenin Cateninbeta-1, beta-catenin, CTNNB, CTNB1 NP_001091679 NP_001091680 NP_001895TCF4 Transcription factor 4, E2-2, ITF2, SEF2, SEF2- AAI25085 1,SEF2-1A, SEF2-1B NP_001077431 NP_003190 EAW63024 EAW63023 EAW63022EAW63021 EAW63020 EAW63019 EAW63018 EAW63017 AAI25086 Q9NQB0 p38Mitogen-activated p38alpha (MAPK14, CSBP2, Crk1, Csbp1, NP_002736.3protein kinase MGC102436, Mxi2, PRKM14, PRKM15, p38, NP_620407p38-alpha, p38MAPK, p38a, p38alpha, OTTMUSP00000021706; cytokinesuppressive anti-inflammatory drug binding protein 1; mitogen activatedprotein kinase 14; p38 MAP kinase alpha; p38 MAPK; p38 alpha; tRNAsynthetase cofactor p38) p38beta (MAPK11, DKFZp586C1322, P38b, Prkm11,Sapk2, Sapk2b, p38-2, p38beta2, mitogen activated protein kinase 11;protein kinase, mitogen activated kinase, 11, p38beta) p38delta (MAPK13,SAPK4, Serk4, OTTMUSP00000028863; SAPK/Erk/kinase 4; mitogen activatedprotein kinase 13; p38 delta MAP kinase) p38gamma (MAPK12, AW123708,Erk6, Prkm12, Sapk3, mitogen activated protein kinase 12; stressactivated protein kinase 3) Epidermal growth EGFR (ERBB, ERBB1, HER1,PIG61, AAG35789 factor receptor (EGFR) mENA, avian erythroblasticleukemia viral (v- NP_005219 erb-b) oncogene homolog; cell growthinhibiting NP_958439 protein 40; cell proliferation-inducing protein 61;NP_958440 epidermal growth factor receptor) NP_958441 ErbB2 (CD340,HER-2, HER-2/neu, HER2, NEU, NGL, TKR1, c-erb B2/neu protein; erbB-2;herstatin; neuroblastoma/glioblastoma derived oncogene homolog; v-erb-b2avian erythroblastic leukemia viral oncogene homolog 2(neuro/glioblastoma derived oncogene homolog)) ErbB3 (ErbB-3, HER3,LCCS2, MDA-BF-1, MGC88033, c-erbB-3, c-erbB3, erbB3-S, p180- ErbB3,p45-sErbB3, p85-sErbB3, erbB-3; lethal congenital contracture syndrome2; v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3)ErbB4 (HER4, MGC138404, p180erbB4, avian erythroblastic leukemia viral(v-erb-b2) oncogene homolog 4; receptor tyrosine-protein kinase erbB- 4;tyrosine kinase-type cell surface receptor HER4; v-erb-a avianerythroblastic leukemia viral oncogene homolog-like 4; v-erb-aerythroblastic leukemia viral oncogene homolog 4) Mitogen-activated JunN-terminal kinase (1); JNK; JNK1; PRKM8; NP_003609 protein kinase 8(JNK) SAPK1; JNK1A2; JNK21B1/2; mitogen- CAG38817 activated proteinkinase-8; MAPK8; JNK-46 AAH65516 JNK-2; Jun N-terminal kinase (2);MAPK9; NP_002741 JNK2; SAPK; p54a; JNK2A; JNK2B; PRKM9; NP_620634JNK-55; JNK2BETA; p54aSAPK; JNK2ALPHA NP_620635 JNK-3; Jun N-terminalkinase (3); MAPK10; NP_620637 JNK3; JNK3A; PRKM10; p493F12; FLJ12099;FLJ33785; MGC50974; p54bSAPK Calcium/calmodulin- CAMKKA; CAMKK alphaprotein; CaM-KK NP_115670 dependent protein alpha; CaM-kinase IV kinase;CaM-kinase kinase NP_757343 kinase beta alpha; CaMKK 1; CaMKK alpha;NP_757344 (CaMKbeta) calcium/calmodulin-dependent protein kinase kinasealpha; calcium/calmodulin-dependent protein kinase 1 alpha;calcium/calmodulin- dependent protein kinase kinase 1, alpha CAMKKB;CAMKK beta protein; CaM-KK beta; NP_006540 CaM-kinase kinase beta; CaMKKbeta; NP_705719 calcium/calmodulin-dependent protein kinase NP_705720kinase beta; NP_757363 calcium/calmodulin-dependent protein kinaseNP_757364 beta; NP_757365 calcium/calmodulin-dependent protein kinaseNP_757380 kinase 2, beta; calcium/calmodulin-dependent protein kinasekinase 2, beta dual leucine zipper- DLK, DLK1, fetal antigen 1 (FA1),PG2, PREF-1, NP_003827 bearing kinase PREF1, ZOG, delta-lika proteindlk, pG2, ABC26857 preadipocyte factor 1 EAW81713 EAW81712 EAW81711AAH14015 AAH13197 AAH07741 P80370 Phosphoinositide 3 PI3-kinase, PI3K,Phosphotidylinositol 3 kinase CAA74194 kinase Cyclin-dependent Cdk5NP_004926 kinase 5 Adenylyl cyclase Adenylyl cyclase 1 (ADCY1);3′,5′-cyclic AMP NM_021116 synthetase (1); ATP pyrophosphate-lyases (1);Ca²⁺/calmodulin-activated adenylyl cyclase; Adenylate cyclase type I;Brain adenylate cyclase 1 Adenylyl cyclase 2 (ADCY2); 3′,5′-cyclic AMPNP_065433 synthetase (2); ATP pyrophosphate-lyase (2); adenylate cyclasetype II; adenylate cyclase 2 (brain); Adenylate cyclase II; Type IIadenylate cyclase Adenylyl cyclase 8 (ADCY8); ATP NP_001106pyrophosphate-lyase 8; adenylate cyclase type VIII; adenylyl cyclase 8;Ca(2+)/calmodulin- activated adenylyl cyclase; adenylate cyclase 8(brain); adenylyl cyclase-8, brain adenylate cyclase 3 (Adcy3, AC3,mKIAA0511, adenylyl cyclase 3) adenylate cyclase 4 (Adcy4, KIAA4004,mKIAA4004) adenylate cyclase 5 (Adcy5, AW121902, Ac5) adenylate cyclase6 (Adcy6, mKIAA0422) adenylate cyclase 7 (Adcy7, AA407758, MGC141539,adenylyl cyclase type VII) adenylate cyclase 9 (Adcy9, AW125421,D16Wsu65e, mKIAA0520) adenylate cyclase 10 (Adcy10, 4930431D04Rik,4931412F17, Sacy, sAC, OTTMUSP00000023839; soluble adenylyl cyclase;testicular soluble adenylyl cyclase) Ih Channel HCN1; hyperpolarizationactivated cyclic NP_066550 nucleotide-gated potassium channel 1; braincyclic nucleotide-gated channel 1; BCNG-1; HAC-2 NP_001185 HCN2;hyperpolarization activated cyclic nucleotide-gated potassium channel 2;brain cyclic nucleotide-gated channel 2; BCNG-2; HAC-1 HCN3; KIAA1535;MGC131493; NP_065948 OTTHUMP00000034062; hyperpolarization activatedcyclic nucleotide-gated potassium channel 3; potassium/sodiumhyperpolarization- activated cyclic nucleotide-gated channel 3 NP_005468HCN4; hyperpolarization activated cyclic nucleotide-gated potassiumchannel 4 BCL2-associated X apoptosis regulator BAX; BCL2L4 Q07812protein (Bax) NP_004315 NP_620116 NP_620118 NP_620119 NP_620120Transcription Actinomycin D

“Isolated” when used to describe the various proteins disclosed herein,means a protein that has been identified and separated and/or recoveredfrom a component of its natural environment. Contaminant components ofits natural environment are materials that may interfere with uses(e.g., uses in therapy or antibody production) for the protein, and mayinclude enzymes, hormones, and other proteinaceous or non-proteinaceoussolutes. In various embodiments, the protein will be purified (i) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, and/or(ii) to homogeneity by SDS-PAGE under non-reducing or reducingconditions using Coomassie blue or silver stain, and/or (iii) tohomogeneity by mass spectroscopic or peptide mapping techniques.Isolated protein includes protein in situ within recombinant cells, asat least one component of the natural environment of the protein inquestion will not be present. Ordinarily, however, isolated protein willbe prepared by at least one purification step. Isolated target proteinsas described herein (or fragments thereof) can be used to makeantibodies as described herein against the target proteins.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid in question. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecules as they exist in natural cells. However, an isolated nucleicacid molecule includes nucleic acid molecules contained in cells thatordinarily express such nucleic acid molecule where, for example, thenucleic acid molecule is in a chromosomal location different from thatof natural cells. An example of an isolated nucleic acid molecule is onelacking 5′ and/or 3′ flanking sequences with which it is contiguous in anatural setting.

As used herein, the terms “antagonist” and “inhibitor” refer to agentscapable of blocking, neutralizing, inhibiting, abrogating, reducingand/or interfering with one or more of the activities of targets and/orreducing the expression of one or more target proteins (or theexpression of nucleic acids encoding one or more target proteins) asdescribed herein. They include, for example, antibodies, polypeptides,peptides, nucleic acid molecules, short interfering RNAs (siRNAs) andother inhibitory RNAs, small molecules (e.g., small inorganicmolecules), polysaccharides, polynucleotides, aptamers, and peptibodies.Antagonists or inhibitors of particular targets as described herein(i.e., targets other than adenylyl cyclase) generally inhibit ordecrease axon degeneration (e.g., by at least 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even100% decrease compared to a control that is untreated with theinhibitor), as described herein. An inhibitor may decrease the activityand/or expression of a target protein by at least 10% (e.g., by at least15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or even 100% decrease) as compared to the expression and/oractivity of the target protein that is untreated with the inhibitor. A“DLK signaling inhibitor” is an agent capable of decreasing the activity(e.g., kinase activity) or the expression of a DLK protein (or a nucleicacid encoding a DLK protein) and/or deceasing the activity and/orexpression of one or more proteins involved in a DLK signaling pathway(e.g., JNK1, JNK2, JNK3, cJun (e.g., cJun-63 and cJun-73), MKK4, andMKK7). Examples of DLK signaling inhibitors include siRNA molecules thatdecrease the expression of a nucleic acid encoding DLK (e.g., desirably,a sequence of GCACTGAATTGGACAACTCTT (SEQ ID NO: 1),GAGTTGTCCAATTCAGTGCTT (SEQ ID NO: 2), GGACATCGCCTCCGCTGA TTT (SEQ ID NO:3), or ATCAGCGGAGGCGATGTCCTT (SEQ ID NO: 4), or GCAAGACCCGTCACCGAAATT(SEQ ID NO: 5), TTTCGGTGACGGG TCTTGCTT (SEQ ID NO: 6),GCGGTGTCCTGGTCTACTATT (SEQ ID NO: 7), or TAGTAGACCAGGACACCGCTT (SEQ IDNO: 8)), JNK1 (e.g., a sequence targeting the JNK1 sequence ofTTGGATGAAGCCATTAGACTA (SEQ ID NO: 9)), JNK2 (e.g., a sequence targetingthe JNK2 sequence of ACCTTTAATGGACAA CATTAA (SEQ ID NO: 10) orAAGGATTAGCTTTGTATCATA (SEQ ID NO: 11)), JNK3 (e.g., a sequence targetingthe JNK3 sequence of CCCGCATGTGTCT GTATTCAA (SEQ ID NO: 12)), cJun(e.g., cJun-63 and cJun-73), MKK4, and MKK7. Additional examples of DLKinhibitors include antibodies that bind to a DLK protein (e.g.,antibodies that recognize unphosphorylated or phosphorylated DLK, suchas the 317, 318, 319, 320, 321, and 322 antibodies described herein),JNK1, JNK2, JNK3, cJun (e.g., cJun-63 and cJun-73), MKK4, and/or MKK7;inhibitors of JNK activity (e.g., SC-202673, SY-CC-401, SP600125, JNKVinhibitor, JNKVIII inhibitor, AS601245, and XG-102, as well as CatalogNos. 420119, 420130, 420131, 420123, 420116, 420118, 420136, 420129,420135, 420134, 420133, 420140, and 420128 from EMD Biosciences);inhibitors of MKK4 activity (e.g., myricetin and the inhibitorsdescribed in WO 04/058764), and inhibitors of MKK7 activity (e.g.,inhibitors described in U.S. Pat. No. 7,195,894 and WO 04/002532). A DLKinhibitor may also be a dominant negative form or kinase-dead form ofDLK protein (or a nucleic acid encoding a dominant negative form or akinase-dead form of DLK protein), such as T278A DLK, S281A DLK, S152ADLK, and the leucine zipper domain of DLK.

Another example of an inhibitor is a “GSK3β inhibitor.” GSK3β inhibitorrefers to an agent capable of decreasing the activity and/or expressionof GSK3β (or a nucleic acid encoding GSK3β) and/or decreasing theactivity and/or the expression of one or more proteins (or a nucleicacid encoding the one or more proteins) that activate GSK3β or theexpression or activity of one or more substrates of GSK3β. Non-limitingexamples of GSK3β inhibitors include SB415286, GSK3β inhibitor I, GSK3βinhibitor VII, GSK3β inhibitor VIII, GSK3β inhibitor XII, and lithiumchloride.

An additional example of an inhibitor is a “G-protein inhibitor.” AG-protein inhibitor refers to an agent capable of decreasing theactivity and/or expression of one or more G-proteins or G-proteincoupled receptors (GPCRs) (or the expression of one or more nucleicacids encoding a G-protein or a GPCR), and/or decreasing the activityand/or expression of one or more proteins downstream of a G-protein or aGPCR. Non-limiting examples of a G-protein inhibitor include siRNAmolecules that decrease the level of expression of a nucleic acidencoding a G-protein or a GPCR, an antibody or peptibody that binds to aG-protein or GPCR, or a small molecule or peptide that inhibits theactivity of a G-protein or GPCR (e.g., SCH202676 and pertussis toxin).

Another example of an inhibitor is a “EGFR pathway inhibitor.” EGFRpathway inhibitor refers to an agent capable of decreasing the activityand/or expression of EGFR protein (or a nucleic acid encoding EGFR)and/or decreasing the activity and/or the expression of one or moreproteins that function downstream of EGFR in the cell (e.g., p38 MAPK,CAMKK, and JNK). Non-limiting examples of EGFR pathway inhibitorsinclude inhibitors of EGFR (e.g., erlotinib, tyrphostin B44, tyrphostinB42/AG 490, AG555, AG494, and PD168393), inhibitors of p38 MAPK (e.g.,SB203580, SB239063, SB202190, and SB239069), inhibitors of CAMKK (e.g.,STO-609), and inhibitors of JNK (e.g., SP600125). Additional examples ofEGFR pathway inhibitors include antibodies and peptibodies that bind toEGFR, p38 MAPK, CAMKK, and/or JNK; and siRNA molecules that decrease theexpression of one or more nucleic acids that encode a protein thatfunctions downstream of EGFR in the cell (e.g., EGFR, p38 MAPK, CAMKK,and/or JNK).

An additional example of an inhibitor is a “CAMKβ inhibitor.” A CAMKβinhibitor refers to an agent capable of decreasing the activity and/orexpression of CAMKβ protein (or a nucleic acid encoding CAMKβ) and/ordecreasing the activity and/or expression of one or more proteins thatfunction downstream of CAMKβ in the cell. Non-limiting examples of CAMKβinhibitors include antibodies and peptibodies that specifically bind toCAMKβ, and siRNA molecules that decrease the expression of one or morenucleic acids that encode CAMKβ or a protein that functions downstreamof CAMKβ.

Another example of an inhibitor is a “cdk5 inhibitor.” A cdk5 inhibitorrefers to an agent capable of decreasing the activity and/or expressionof cdk5 protein (or a nucleic acid encoding cdk5) and/or decreasing theactivity and/or expression of one or more proteins that functiondownstream of cdk5 in the cell. Non-limiting examples of cdk5 inhibitorsinclude antibodies and peptibodies that specifically bind to cdk5, andsiRNA molecules that decrease the expression of one or more nucleicacids that encode cdk5 or a protein that functions downstream of cdk5.

An additional example of an inhibitor is a “TCF4 inhibitor.” A TCF4inhibitor refers to an agent capable of decreasing the activity and/orexpression of TCF4 protein (or a nucleic acid encoding TCF4) and/ordecreasing the activity and/or expression of a gene regulated by TCF4protein. Non-limiting examples of TCF4 inhibitors include antibodies andpeptibodies that specifically bind to TCF4 or a protein encoded by agene regulated by TCF4, and siRNA molecules that decrease the expressionof one or more nucleic acids that encode TCF4 or decrease the expressionof an mRNA encoded by a gene regulated by TCF4.

An additional example of an inhibitor is a “β-catenin inhibitor.” Aβ-catenin refers to an agent capable of decreasing the activity and/orexpression of “β-catenin protein (or a nucleic acid encoding “β-catenin)and/or decreasing the activity and/or expression of a gene regulated by“β-catenin protein. Non-limiting examples of β-catenin inhibitorsinclude antibodies and peptibodies that specifically bind to β-cateninor a protein encoded by a gene regulated by β-catenin, and siRNAmolecules that decrease the expression of one or more nucleic acids thatencode β-catenin or decrease the expression of an mRNA encoded by a generegulated by β-catenin.

An additional example of an inhibitor is an “adenyl cyclase inhibitor.”An adenyl cyclase inhibitor refers to an agent capable of decreasing theactivity and/or expression of adenyl cyclase protein (or a nucleic acidencoding adenyl cyclase) and/or decreasing the activity and/orexpression of one or more proteins that function downstream of adenylcyclase in the cell. Non-limiting examples of adenyl cyclase inhibitorsinclude antibodies and peptibodies that specifically bind to adenylcyclase, and siRNA molecules that decrease the expression of one or morenucleic acids that encode adenyl cyclase or a protein that functionsdownstream of adenyl cyclase. Additional examples of adenyl cyclaseinhibitors include small molecules that inhibit the activity of adenylcyclase (e.g., forksolin and NKH 477).

The terms “agonist” or “activator” as used herein refer to agentscapable of increasing or activating one or more of the activities oftargets as described herein, and include, for example, antibodies,polypeptides, peptides, nucleic acid molecules, short interfering RNAs(siRNAs) or other inhibitory RNAs, small molecules (e.g., smallinorganic molecules), polysaccharides, polynucleotides, aptamers, andpeptibodies. Agonists or activators of adenylyl cyclase as describedherein generally inhibit or decrease axon degeneration, while agonistsor activators of the other particular targets described herein can beconsidered to activate axon degeneration.

The term “antibody” herein is used in the broadest sense understood inthe art and specifically covers, for example, intact antibodies,monoclonal antibodies, polyclonal antibodies, monospecific antibodies,multispecific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, antibody fragments, provided that theyexhibit the desired biological activity, and intrabodies.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site or epitope. Furthermore, in contrast topolyclonal antibody preparations, which include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on an antigen. Inaddition to their specificity, monoclonal antibodies are advantageous inthat they may be synthesized so that they are uncontaminated by otherantibodies. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature,256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using, for example, thetechniques described in Clackson et al., Nature 352:624-628, 1991, andMarks et al., J. Mol. Biol. 222:581-597, 1991.

Antibodies specifically include “chimeric” antibodies in which a portionof the heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, provided that they exhibit the desired biologicalactivity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.Acad. Sci. U.S.A. 81:6851-6855, 1984). Chimeric antibodies of interestherein include primatized antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g., OldWorld Monkey, Ape, etc.) and human constant region sequences.

“Antibody fragments” comprise a portion of an intact antibody, such asthe antigen-binding or variable region thereof. Examples of antibodyfragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies;linear antibodies; and single-chain antibody molecules.

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody comprising a heavy chain variable domain(V_(H)) and a light chain variable domain (V_(L)), where the V_(H)V_(L)unit has polyepitopic specificity (i.e., is capable of binding to morethan one different epitope on one or more biological molecules). If themultispecific antibody binds to two epitopes, it can be designated as a“bispecific antibody.” Multispecific antibodies include, but are notlimited to, full length antibodies, antibodies having two or more V_(L)and V_(H) domains, antibody fragments such as Fab, Fv, dsFv, scFv,diabodies, bispecific diabodies and triabodies, antibody fragments thathave been linked covalently or non-covalently. “Polyepitopicspecificity” refers to the ability to specifically bind to two or moredifferent epitopes on the same or different target(s).

An “intact” antibody is one that comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H)1, C_(H)2, and C_(H)3. The constant domains maybe native sequence constant domains (e.g., human native sequenceconstant domains) or amino acid sequence variants thereof. In oneexample, the intact antibody has one or more effector functions.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit, or nonhuman primate having the desired specificityand affinity. In some instances, framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains (Fab, Fab′, F(ab′)2,Fabc, Fv), in which all or substantially all of the hypervariable loopscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. For further details see, for example, Jones etal., Nature 321:522-525, 1986; Riechmann et al., Nature 332:323-329,1988; and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992.

The term “hypervariable region” when used herein refers to the regionsof an antibody variable domain that are hypervariable in sequence and/orform structurally defined loops. The hypervariable region comprisesamino acid residues from a “complementarity determining region” or “CDR”(i.e., residues 24-34, 50-56, and 89-97 in the light chain variabledomain and 31-35, 50-65, and 95-102 in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991) and/or those residues from a “hypervariable loop” (i.e., residues26-32, 50-52, and 91-96 in the light chain variable domain and 26-32,53-55, and 96-101 in the heavy chain variable domain; Chothia et al., J.Mol. Biol. 196:901-917, 1987). In both cases, the variable domainresidues are numbered according to Kabat et al., supra, as discussed inmore detail below. “Framework” or “FR” residues are those variabledomain residues other than the residues in the hypervariable regions asherein defined.

A “parent antibody” or “wild-type” antibody is an antibody comprising anamino acid sequence that lacks one or more amino acid sequencealterations compared to an antibody variant as herein disclosed. Thus,the parent antibody generally has at least one hypervariable region thatdiffers in amino acid sequence from the amino acid sequence of thecorresponding hypervariable region of an antibody variant. The parentpolypeptide may comprise a native sequence (i.e., a naturally occurring)antibody (including a naturally occurring allelic variant), or anantibody with pre-existing amino acid sequence modifications (such asinsertions, deletions, and/or other alterations) of a naturallyoccurring sequence. The terms “wild type,” “WT,” “wt,” and “parent” or“parental” antibody may be used interchangeably.

As used herein, “antibody variant” or “variant antibody” refers to anantibody that has an amino acid sequence that differs from the aminoacid sequence of a parent antibody. In one example, the antibody variantcomprises a heavy chain variable domain or a light chain variable domainhaving an amino acid sequence that is not found in nature. Such variantsnecessarily have less than 100% sequence identity or similarity with theparent antibody. In one embodiment, the antibody variant will have anamino acid sequence from about 75% to less than 100% amino acid sequenceidentity or similarity with the amino acid sequence of either the heavyor light chain variable domain of the parent antibody, for example,about 80% to less than 100%, from about 85% to less than 100%, fromabout 90% to less than 100%, or from about 95% to less than 100%. Theantibody variant is generally one that comprises one or more amino acidalterations in or adjacent to one or more hypervariable regions thereof.

An “amino acid alteration” refers to a change in the amino acid sequenceof a predetermined amino acid sequence. Exemplary alterations includeinsertions, substitutions, and deletions. An “amino acid substitution”refers to the replacement of an existing amino acid residue in apredetermined amino acid sequence with another, different amino acidresidue.

A “replacement” amino acid residue refers to an amino acid residue thatreplaces or substitutes another amino acid residue in an amino acidsequence. The replacement residue may be a naturally occurring ornon-naturally occurring amino acid residue.

An “amino acid insertion” refers to the introduction of one or moreamino acid residues into a predetermined amino acid sequence. The aminoacid insertion may comprise a “peptide insertion” in which case apeptide comprising two or more amino acid residues joined by peptidebond(s) is introduced into the predetermined amino acid sequence. Wherethe amino acid insertion involves insertion of a peptide, the insertedpeptide may be generated by random mutagenesis such that it has an aminoacid sequence that does not exist in nature. An amino acid alteration“adjacent a hypervariable region” refers to the introduction orsubstitution of one or more amino acid residues at the N-terminal and/orC-terminal end of a hypervariable region, such that at least one of theinserted or replacement amino acid residue(s) forms a peptide bond withthe N-terminal or C-terminal amino acid residue of the hypervariableregion in question.

A “naturally occurring amino acid residue” is one encoded by the geneticcode, generally selected from the group consisting of: alanine (Ala);arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys);glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His);isoleucine (Ile): leucine (Leu); lysine (Lys); methionine (Met);phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr);tryptophan (Trp); tyrosine (Tyr); and valine (Val).

A “non-naturally occurring amino acid residue” as referred to herein isan amino acid residue other than those naturally occurring amino acidresidues listed above, which is able to covalently bind adjacent aminoacid residue(s) in a polypeptide chain. Examples of non-naturallyoccurring amino acid residues include norleucine, ornithine, norvaline,homoserine, and other amino acid residue analogues such as thosedescribed in Ellman et al., Meth. Enzym. 202:301-336, 1991. To generatesuch non-naturally occurring amino acid residues, the procedures ofNoren et al., Science 244:182, 1989, and Ellman et al., supra, can beused. Briefly, these procedures involve chemically activating asuppressor tRNA with a non-naturally occurring amino acid residuefollowed by in vitro transcription and translation of the RNA.

Throughout this disclosure, reference is made to the numbering systemfrom Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md., 1987 and 1991).In these compendiums, Kabat lists many amino acid sequences forantibodies for each subclass, and lists the most commonly occurringamino acid for each residue position in that subclass. Kabat uses amethod for assigning a residue number to each amino acid in a listedsequence, and this method for assigning residue numbers has becomestandard in the field. The Kabat numbering scheme is followed in thisdescription. For purposes of this invention, to assign residue numbersto a candidate antibody amino acid sequence that is not included in theKabat compendium, one follows the following steps. Generally, thecandidate sequence is aligned with any immunoglobulin sequence or anyconsensus sequence in Kabat. Alignment may be done by hand or bycomputer using commonly accepted computer programs, such as the Align 2program. Alignment may be facilitated by using some amino acid residuesthat are common to most Fab sequences. For example, the light and heavychains each typically have two cysteines that have the same residuenumbers; in V_(L) domain the two cysteines are typically at residuenumbers 23 and 88, and in the V_(H) domain the two cysteine residues aretypically at numbers 22 and 92. Framework residues generally, but notalways, have approximately the same number of residues, however the CDRswill vary in size. For example, in the case of a CDR from a candidatesequence that is longer than the CDR in the sequence in Kabat to whichit is aligned, typically suffixes are added to the residue number toindicate the insertion of additional residues. For candidate sequencesthat, for example, align with a Kabat sequence for residues 34 and 36but have no residue between them to align with residue 35, the number 35is simply not assigned to a residue.

As used herein, an antibody with a “high-affinity” is an antibody havinga K_(D), or dissociation constant, in the nanomolar (nM) range orbetter. A K_(D) in the “nanomolar range or better” may be denoted by XnM, where X is a number less than about 10.

The term “filamentous phage” refers to a viral particle capable ofdisplaying a heterogenous polypeptide on its surface and includes,without limitation, f1, fd, Pf1, and M13. The filamentous phage maycontain a selectable marker such as tetracycline (e.g., “fd-tet”).Various filamentous phage display systems are well known to those ofskill in the art (see, e.g., Zacher et al., Gene 9:127-140, 1980, Smithet al., Science 228:1315-1317, 1985; and Parmley et al., Gene73:305-318, 1988).

The term “panning” is used to refer to the multiple rounds of ascreening process that is used in the identification and isolation ofphages carrying compounds, such as antibodies, with high affinity andspecificity to a target.

The term “short-interfering RNA (siRNA)” refers to small double-strandedRNAs that interfere with gene expression. siRNAs are mediators of RNAinterference, the process by which double-stranded RNA silenceshomologous genes. siRNAs typically are comprised of two single-strandedRNAs of about 15-25 nucleotides in length that form a duplex, which mayinclude single-stranded overhang(s). Processing of the double-strandedRNA by an enzymatic complex, for example, polymerases, results incleavage of the double-stranded RNA to produce siRNAs. The antisensestrand of the siRNA is used by an RNA interference (RNAi) silencingcomplex to guide mRNA cleavage, thereby promoting mRNA degradation. Tosilence a specific gene using siRNAs, for example, in a mammalian cell,a base pairing region is selected to avoid chance complementarity to anunrelated mRNA. RNAi silencing complexes have been identified in theart, such as, for example, by Fire et al., Nature 391:806-811, 1998, andMcManus et al., Nat. Rev. Genet. 3(10):737-747, 2002.

The term “interfering RNA (RNAi)” is used herein to refer to adouble-stranded RNA that results in catalytic degradation of specificmRNAs, and thus can be used to inhibit/lower expression of a particulargene.

As used herein, the term “disorder” in general refers to any conditionthat would benefit from treatment with the agents or inhibitors of thepresent invention, including any disease or disorder that can be treatedby effective amounts of inhibitors of the targets described herein (oractivators, in the case of adenylyl cyclase). Non-limiting examples ofdisorders to be treated herein include those listed in section E of thepresent application, below.

The terms “treating,” “treatment,” and “therapy” as used herein refer tocurative therapy, prophylactic therapy, and preventative therapy.Consecutive treatment or administration refers to treatment on at leasta daily basis without interruption in treatment by one or more days.Intermittent treatment or administration, or treatment or administrationin an intermittent fashion, refers to treatment that is not consecutive,but rather cyclic in nature. Treatment according to the methods of theinvention can result in complete relief or cure from a disease orcondition, or partial amelioration of one or more symptoms of thedisease or condition, and can be temporary or permanent.

The phrases “preventing axon degeneration,” “preventing neurondegeneration,” “inhibiting axon degeneration,” or “inhibiting neurondegeneration” as used herein include (i) the ability to inhibit orprevent axon or neuron degeneration in patients newly diagnosed ashaving a neurodegenerative disease or at risk of developing a newneurodegenerative disease and (ii) the ability to inhibit or preventfurther axon or neuron degeneration in patients who are alreadysuffering from, or have symptoms of, a neurodegenerative disease.Preventing axon or neuron degeneration includes decreasing or inhibitingaxon or neuron degeneration, which may be characterized by complete orpartial inhibition of neuron or axon degeneration. This can be assessed,for example, by analysis of neurological function. The above-listedterms also include in vitro and ex vivo methods. Further, the phrases“preventing neuron degeneration” and “inhibiting neuron degeneration”include such inhibition with respect to the entire neuron or a portionthereof, such as the neuron cell body, axons, and dendrites. Theadministration of one or more agent as described herein may result in atleast a 10% decrease (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% decrease) inone or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) symptoms of a disorderof the nervous system; a condition of the nervous system that issecondary to a disease, condition, or therapy having a primary effectoutside of the nervous system; an injury to the nervous system caused byphysical, mechanical, or chemical trauma, pain; an ocular-relatedneurodegeneration; memory loss; or a psychiatric disorder (e.g.,tremors, slowness of movement, ataxia, loss of balance, depression,decreased cognitive function, short-term memory loss, long-term memoryloss, confusion, changes in personality, language difficulties, loss ofsensory perception, sensitivity to touch, numbness in extremities,muscle weakness, muscle paralysis, muscle cramps, muscle spasms,significant changes in eating habits, excessive fear or worry, insomnia,delusions, hallucinations, fatigue, back pain, chest pain, digestiveproblems, headache, rapid heart rate, dizziness, blurred vision, shadowsor missing areas of vision, metamorphopsia, impairment in color vision,decreased recovery of visual function after exposure to bright light,and loss in visual contrast sensitivity) in a subject or populationcompared to a control subject or population that does not receive theone or more agent described herein. The administration of one or moreagent as described herein may result in at least a 10% decrease (e.g.,at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or even 100% decrease) in the number of neurons(or neuron bodies, axons, or dendrites thereof) that degenerate in aneuron population or in a subject compared to the number of neurons (orneuron bodies, axons, or dendrites thereof) that degenerate in neuronpopulation or in a subject that is not administered the one or more ofthe agents described herein. The administration of one or more agent asdescribed herein may result in at least a 10% decrease (e.g., at least15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or even 100% decrease) in the likelihood of developing adisorder of the nervous system; a condition of the nervous system thatis secondary to a disease, condition, or therapy having a primary effectoutside of the nervous system; an injury to the nervous system caused byphysical, mechanical, or chemical trauma, pain; an ocular-relatedneurodegeneration; memory loss; or a psychiatric disorder in a subjector a subject population compared to a control subject or population nottreated with the one or more agent described herein.

The term “administering” as used herein refers to contacting a neuron orportion thereof with an inhibitor as described herein. This includesadministration of the inhibitor to a subject in which the neuron orportion thereof is present, as well as introducing the inhibitor into amedium in which a neuron or portion thereof is cultured.

The term “neuron” as used herein denotes nervous system cells thatinclude a central cell body or soma, and two types of extensions orprojections: dendrites, by which, in general, the majority of neuronalsignals are conveyed to the cell body, and axons, by which, in general,the majority of neuronal signals are conveyed from the cell body toeffector cells, such as target neurons or muscle. Neurons can conveyinformation from tissues and organs into the central nervous system(afferent or sensory neurons) and transmit signals from the centralnervous systems to effector cells (efferent or motor neurons). Otherneurons, designated interneurons, connect neurons within the centralnervous system (the brain and spinal column). Certain specific examplesof neuron types that may be subject to treatment according to theinvention include cerebellar granule neurons, dorsal root ganglionneurons, and cortical neurons.

The term “mammal” as used herein refers to any animal classified as amammal, including humans, higher non-human primates, rodents, anddomestic and farm animals, such as cows, horses, dogs, and cats. In oneembodiment of the invention, the mammal is a human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutiveadministration, in any order.

An “effective amount” is an amount sufficient to effect beneficial ordesired therapeutic (including preventative) results. An effectiveamount can be administered in one or more administrations.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. The term “progeny” refers to any and all offspring of everygeneration subsequent to an originally transformed cell or cell line.Mutant progeny that have the same function or biological activity asscreened for in the originally transformed cell are included.

A “small molecule” is defined herein to have a molecular weight belowabout 1000 Daltons, for example, below about 500 Daltons. Smallmolecules may be organic or inorganic, and may be isolated from, forexample, compound libraries or natural sources, or may be obtained byderivatization of known compounds.

“Aptamers” are nucleic acid molecules that form tertiary structures thatspecifically bind to a target molecule, such as the targets describedherein. The generation and therapeutic use of aptamers are wellestablished in the art (see, e.g., U.S. Pat. No. 5,475,096). Aptamersused in the invention can include modified nucleotides (e.g., nucleicacid analogs or derivatives) that are stable from degradation in vivo.At a minimum, the nucleic acid molecules are designed to be sufficientlystable in vivo, for a sufficient length of time, to allow therapeuticaction to take place prior to degradation and/or elimination. Asnon-limiting examples, such nucleotide analogs can be selected from thegroup consisting of phosphorothioate, boranophosphate,methyl-phosphonate, and 2′-O-methyl analogs, and analogs thereof. As aspecific example, the analog can be 2′-deoxy-2′-fluoro-RNA (2′-F-RNA).

“Peptibodies” are peptide sequences linked to an amino acid sequenceencoding a fragment or portion of an immunoglobulin molecule. Thepeptide sequences may be derived from randomized sequences selected byany method for specific binding, including but not limited to, phagedisplay technology. In one embodiment, the selected polypeptide may belinked to an amino acid sequence encoding the Fc portion of animmunoglobulin. Peptibodies that specifically bind to and modulate thetargets described herein, leading to inhibition of neuron degeneration,are also useful in the methods of the invention.

The term “pharmaceutically acceptable salt” is used herein to refer tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.describe pharmaceutically acceptable salts in detail in J. Pharm. Sci.66:1-19, 1977. The salts can be prepared in situ during the finalisolation and purification of the compounds of the invention orseparately by reacting the free base group with a suitable organic acid.Representative acid addition salts include acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphersulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts andthe like. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamineand the like.

B. Screening Assays to Identify and Characterize Inhibitors of NeuronDegeneration

The invention is based in part on the discovery that certain modulatorsof the target proteins and activities listed in Table 1 in section A,above (dual leucine zipper-bearing kinase (DLK), glycogen synthasekinase 3β (GSK3β), p38 mitogen-activated protein kinase (p38 MAPK),epidermal growth factor receptor (EGFR), phosphoinositide 3 kinase(PI3K), cyclin-dependent kinase 5 (Cdk5), adenylyl cyclase, c-JunN-terminal kinase (JNK), BCL2-associated X protein (Bax), Ih channel,calcium/calmodulin-dependent protein kinase kinase (CaMKK), a G-protein,a G-protein coupled receptor, transcription factor 4 (TCF4), β-catenin,transcription, and protein synthesis) are effective inhibitors of neuron(such as axon) degeneration. The modulators function as inhibitors ofthe target proteins and activities, with the exception of the modulatorof adenylyl cyclase, which is an activator. The inhibitors of neuron oraxon degeneration are referred to as “inhibitors” herein, regardless oftheir effects on their respective targets, as they are inhibitors ofneuron or axon degeneration. They are also referred to herein as“agents” that modulate the activity of a target protein or activity inthe neuron or axon, leading to inhibition of neuron or axondegeneration.

The invention includes methods of inhibiting neuron or axon degenerationby use of inhibitors as described herein. As described in further detailbelow, the methods can be carried out in vivo, such as in the treatmentof neurological disorders or injuries to the nervous system. The methodscan also be carried out in vitro or ex vivo, such as in laboratorystudies of neuron function and in the treatment of nerve grafts ortransplants. These methods are described further below, after adescription of methods for identifying and testing inhibitors used inthe invention.

Inhibitors that can be used in the invention include those listed inTable 2 (section C, below), which have been shown in assays describedherein to prevent neuron or axon degeneration, as well as additional,known inhibitors of the targets described herein (see, e.g., Table 3).Additional inhibitors for use in the invention can be identified usingstandard screening methods specific for each target, as summarizedbelow. These assays can also be used to confirm the activities ofderivatives of compounds found to have a desired activity, which aredesigned according to standard medicinal chemistry approaches. After aninhibitor (or activator, in the case of adenylyl cyclase) is confirmedas being active with respect to a particular target, the inhibitors canbe tested in models of neuron or axon degeneration, as described herein,as well as in appropriate animal model systems.

Exemplary assays for identifying inhibitors of the targets listed inTable 1, as well as for identifying and characterizing additionalinhibitors of neuron or axon degeneration, which can be used in themethods of the invention, are described briefly as follows.

i) Cell-Based and In Vitro Assays for Inhibitors of Neuron or AxonDegeneration

Assays for confirming that an inhibitor of the targets described hereinalso inhibits neuron or axon degeneration, as well as for identifyingadditional inhibitors of neuron or axon degeneration, are described indetail in the Examples, below, and are briefly summarized as follows.These assays include (i) anti-Nerve Growth Factor (anti-NGF) antibodyassays, (ii) serum deprivation/potassium chloride (KCl) reductionassays, (iii) rotenone degeneration assays, and (iv) vincristinedegeneration assays. Additional assays for assessing neuron or axondegeneration that are known in the art can also be used in theinvention.

NGF is a small, secreted protein involved in differentiation andsurvival of target neurons. Treatment of cultured neurons with NGFresults in proliferation of axons, while treating such neurons withanti-NGF antibodies results in axon degeneration. Treatment of neuronswith anti-NGF antibodies also leads to several different morphologicalchanges that are detectable by microscopy, and which can be monitored toobserve the effects of candidate inhibitors. These changes, which aredescribed further in Example 1 and illustrated in, for example, FIGS.1-4, include varicosity formation, loss of elongated mitochondria,accumulation of mitochondria in varicosities, cytoskeletal disassembly,and axon fragmentation. Agents that are found to counter any of themorphological changes induced by anti-NGF antibodies can be consideredas candidate inhibitors of neuron or axon degeneration, which may, ifdesired, be tested in additional systems, such as those describedherein.

The serum deprivation/KCl reduction assay is based on the use ofcultures of cerebellar granule neurons (CGN) isolated from mouse (e.g.,P7 mouse) brains. In this assay, the neurons are cultured in a mediumincluding KCl and then are switched to medium containing less KCl (BasalMedium Eagles including 5 mM KCl), which induces neuron degeneration.Agents that are found to block or reduce neuron degeneration upon KClwithdrawal, which can be detected by, for example, analysis of images offixed neurons stained with a neuronal marker (e.g., anti-class IIIbeta-tubulin) can be considered as candidate inhibitors of neuron oraxon degeneration, which may, if desired, be tested in additionalsystems, such as those described herein.

Another model of neuron or axon degeneration involves contact ofcultured neurons with rotenone(2R,6aS,12aS)-1,2,6,6a,12,12a-hexahydro-2-isopropenyl-8,9-dimethoxychromeno[3,4-b]furo(2,3-h)chromen-6-one),which is a pesticide and insecticide that naturally occurs in the rootsand stems of several plants, interferes with mitochondrial electrontransport, and causes Parkinson's disease-like symptoms when injectedinto rats. Agents that are found to block or reduce degeneration ofneurons cultured in the presence of rotenone, which can be detected by,for example, analysis of images of fixed neurons stained with, e.g., anantibody against neuron specific beta III tubulin, can be considered ascandidate inhibitors of neuron or axon degeneration, which may, ifdesired, be tested in additional systems, such as those describedherein.

An additional model of neuron or axon degeneration involves contact ofcultured neurons with vincristine, an alkaloid that binds to tubulindimers and prevents assemble of microtubule structures. Agenst that arefound to block or reduce degeneration of neurons cultured in thepresence of vincristine, which can be detected by, for example, analysisof images of fixed neurons stained with, e.g., an antibody againstneuron specific beta III tubulin, can be considered as candidateinhibitors of neuron or axon degeneration, which may, if desired, betested in additional systems, such as those described herein.

In addition to the assays described above, for which the read-out isinhibition of neuron or axon degeneration, the invention also employsassays directed at detecting inhibitors of the targets listed in Table1, for which the read-out is, for example, target binding or targetactivity. Thus, the invention includes the use of screening assays forinhibitors of the targets listed in Table 1, which may be designed toidentify compounds that bind or complex with the targets, or otherwiseinterfere with their activities. The screening assays include assaysamenable to high-throughput screening of chemical libraries, making themsuitable for identifying small molecule drug candidates. Generally,binding assays and activity assays are used. The assays can be performedin a variety of formats, including, without limitation, kinase assays,biochemical screening assays, immunoassays, and cell-based assays, asdetermined to be appropriate, based on the subject target.

In binding assays, the interaction is binding, and the complex formedcan be isolated or detected in the reaction mixture. In a particularembodiment, either the target polypeptide or the drug candidate isimmobilized on a solid phase, e.g., on a microtiter plate, by covalentor non-covalent attachments. Non-covalent attachment generally isaccomplished by coating the solid surface with a solution of thepolypeptide and drying. Alternatively, an immobilized antibody, e.g., amonoclonal antibody, specific for the target polypeptide to beimmobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g., the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g., by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labeledantibody specifically binding the immobilized complex.

If the candidate compound is a polypeptide that interacts with but doesnot bind to the target, the interaction of the target with therespective polypeptide can be assayed by methods well known fordetecting protein-protein interactions. Such assays include traditionalapproaches, such as, e.g., cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers (Fields etal., Nature (London) 340:245-246, 1989; Chien et al., Proc. Natl. Acad.Sci. U.S.A. 88:9578-9582, 1991) as disclosed by Chevray et al., Proc.Natl. Acad. Sci. U.S.A. 89:5789-5793, 1991. Many transcriptionalactivators, such as yeast GAL4, consist of two physically discretemodular domains, one acting as the DNA-binding domain, and the other onefunctioning as the transcription-activation domain. The yeast expressionsystem described in the foregoing publications (generally referred to asthe “two-hybrid system”) takes advantage of this property, and employstwo hybrid proteins, one in which the target protein is fused to theDNA-binding domain of GAL4, and another, in which candidate activatingproteins are fused to the activation domain. The expression of aGAL1-lacZ reporter gene under control of a GAL4-activated promoterdepends on reconstitution of GAL4 activity via protein-proteininteraction. Colonies containing interacting polypeptides are detectedwith a chromogenic substrate for β-galactosidase. A complete kit(MATCHMAKER™) for identifying protein-protein interactions between twospecific proteins using the two-hybrid technique is commerciallyavailable from Clontech. This system can also be extended to map proteindomains involved in specific protein interactions as well as to pinpointamino acid residues that are crucial for these interactions.

Compounds that interfere with the interaction of the target and otherintra- or extracellular components can be tested as follows. Usually areaction mixture is prepared containing the target and the intra- orextracellular component under conditions and for a time allowing for theinteraction of the two products. To test the ability of a candidatecompound to inhibit the interaction of the target, the reaction is runin the absence and in the presence of the test compound. In addition, aplacebo may be added to a third reaction mixture, to serve as a positivecontrol.

Assays for measuring the impact of a candidate inhibitor on the activityof a protein kinase are known in the art, and include directphosphorylation assays, typically interpreted via radio-labeledphosphate, phosphorylation-specific antibodies to a substrate, andcell-based assays that measure the downstream consequence of kinaseactivity, e.g., activation of a reporter gene. Both of these majorstrategies, in addition to alternative assays based on fluorescencepolarization, may be used in small-scale or high-throughput format toidentify, validate, or characterize an inhibitor (see, for example,Favata et al., J. Biol. Chem. 273:18623-18632, 1998; Parker et al., J.Biomol. Screening 5:77-99, 2000; Singh et al., Comb. Chem. HighThroughput Screen 8:319-325, 2005; Garton et al., Meth. Enz.439:491-500, 2008; and Kupchko et al., Anal. Biochem. 317:210-217,2003).

The screening assays specifically discussed herein are for the purposeof illustration only. A variety of other assays, which can be selecteddepending on the particular target and type of antagonist candidatesscreened (e.g., antibodies, polypeptides, peptides, non-peptide smallorganic molecules, nucleic acid molecules, etc.) are well known to thoseskilled in the art and may also be used in the present invention.

The assays described herein may be used to screen libraries of compoundsincluding, without limitation, chemical libraries, natural productlibraries (e.g., collections of microorganisms, animals, plants, etc.),and combinatorial libraries comprised of random peptides,oligonucleotides, or small organic molecules. In a particularembodiment, the assays herein are used to screen antibody librariesincluding, without limitation, naïve human, recombinant, synthetic, andsemi-synthetic antibody libraries. The antibody library can, forexample, be a phage display library, including monovalent libraries,displaying on average one single-chain antibody or antibody fragment perphage particle, and multi-valent libraries, displaying, on average, twoor more antibodies or antibody fragments per viral particle. However,the antibody libraries to be screened in accordance with the presentinvention are not limited to phage display libraries. Other displaytechniques include, for example, ribosome or mRNA display (Mattheakis etal., Proc. Natl. Acad. Sci. U.S.A. 91:9022-9026, 1994; Hanes et al.,Proc. Natl. Acad. Sci. U.S.A. 94:4937-4942, 1997), microbial celldisplay, such as bacterial display (Georgiou et al., Nature Biotech.15:29-34, 1997), or yeast cell display (Kieke et al., Protein Eng.10:1303-1310, 1997), display on mammalian cells, spore display, viraldisplay, such as retroviral display (Urban et al., Nucleic Acids Res.33:e35, 2005), display based on protein-DNA linkage (Odegrip et al.,Proc. Acad. Natl. Sci. U.S.A. 101:2806-2810, 2004; Reiersen et al.,Nucleic Acids Res. 33:e10, 2005), and microbead display (Sepp et al.,FEBS Lett. 532:455-458, 2002).

The results obtained in the primary binding/interaction assays hereincan be confirmed in in vitro and/or in vivo assays of axon degeneration.Alternatively, in vitro and/or in vivo assays of axon degeneration maybe used as primary assays to identify inhibitors and antagonists asdescribed herein.

ii) Animal Models of Neuron or Axon Degeneration

In vivo assays for use in the invention include animal models of variousneurodegenerative diseases, such as animal models of amyotrophic lateralsclerosis (ALS), Alzheimer's disease, Parkinson's disease, and multiplesclerosis (e.g., experimental autoimmune encephalitis (EAE) in mice). Inaddition, spinal cord and traumatic brain injury models can be used.Non-limiting examples of in vivo assays that can be used incharacterizing inhibitors for use in the invention are described asfollows.

In the case of amyotrophic lateral sclerosis (ALS), a transgenic mousethat expresses a mutant form of superoxide dismutase 1 (SOD1)recapitulates the phenotype and pathology of ALS (Rosen et al., Nature362(6415):59-62, 1993). In addition to the SOD1 mouse, several mousemodels of amyotrophic lateral sclerosis (ALS) have been developed andcan be used in the invention. These include motor neuron degeneration(Mnd), progressive motor neuropathy (pmn), wobbler (Bird et al., ActaNeuropathologica 19(1):39-50, 1971), and TDP-43 mutant transgenic mice(Wegorzewska et al., Proc. Natl. Acad. Sci. U.S.A., e-published on Oct.15, 2009). In addition, a canine model has been developed and can beused in the invention (hereditary canine spinal muscular atrophy(HCSMA)).

Animal models that simulate the pathogenic, histological, biochemical,and clinical features of Parkinson's disease, which can be used incharacterizing inhibitors for use in the methods of the presentinvention, include the reserpine (rabbit; Carlsson et al., Nature180:1200, 1957); methamphetamine (rodent and non-human primates; Seidenet al., Drug Alcohol Depend 1:215-219, 1975); 6-OHDA (rat; Perese etal., Brain Res. 494:285-293, 1989); MPTP (mouse and non-human primates;Langston et al., Ann. Neurol. 46:598-605, 1999); paraquat/maneb (mouse;Brooks et al., Brain Res. 823:1-10, 1999 and Takahashi et al., Res.Commun. Chem. Pathol. Pharmacol. 66:167-170, 1989); rotenone (rat;Betarbet et al., Nat. Neurosci. 3:1301-1306, 2000); 3-nitrotyrosine(mouse; Mihm et al., J. Neurosci. 21:RC149, 2001); and mutatedα-synuclein (mouse and Drosophila; Polymeropoulos et al., Science276:2045-2047, 1997) models.

Genetically-modified animals, including mice, flies, fish, and worms,have been used to study the pathogenic mechanisms behind Alzheimer'sdisease. For example, mice transgenic for β-amyloid develop memoryimpairment consistent with Alzheimer's disease (Gotz et al., Mol.Psychiatry 9:664-683, 2004). Models such as these may be used incharacterizing the inhibitors.

Several animal models are used in the art to study stroke, includingmice, rats, gerbils, rabbits, cats, dogs, sheep, pigs, and monkeys. Mostfocal cerebral ischemia models involve occlusion of one major cerebralblood vessel such as the middle cerebral artery (see, e.g., Garcia,Stroke 15:5-14, 1984 and Bose et al., Brain Res. 311:385-391, 1984). Anyof these models may also be used in the invention.

C. Inhibitors of Neuron or Axon Degeneration

As described further below, in the Examples, the compounds listed inTable 2, below, were identified as inhibitors of neuron or axondegeneration. These compounds, as well as other agents (see, e.g., Table3) that inhibit (or activate, in the case of adenylyl cyclase) thetargets and processes listed in Table 2, can be used in methods ofinhibiting neuron or axon degeneration, according to the invention.

TABLE 2 Target I. Compounds (Source) II. Structure Proteasome MG132(Calbiochem Cat. No. 474790) (CAS: 133407-82-6)

GSK3β SB 415286 (Tocris Cat. No. 1617) (CAS: 264218-23-7)

GSK3β inhibitor I (Calbiochem Cat. No. 361540) (CAS: 327036-89-5)

GSK3β inhibitor VII (Calbiochem Cat. No. 361548) (CAS: 99-73-0)

GSK3β inhibitor VII (Calbiochem Cat. No. 361549) (CAS: 487021-52-3)

GSK3β inhibitor XII (Calbiochem Cat. No. 361554) (CAS: 601514-19-6)

Lithium Chloride LiCl Other lithium salts (e.g., lithium carbonate(e.g., Lithobid ™) and lithium citrate) (CAS: 7447-41-8) P38 MAPK SB202190 (Calbiochem Cat. No. 559388) (CAS: 152121-30-7)

SB 239063 (Calbiochem Cat. No. 559404) (CAS: 193551-21-2)

SB 239069 SB 203580 (Calbiochem Cat. No. 559389) (CAS: 152121-47-6)

EGFRK AG 556 (Calbiochem Cat. No. 658415) (CAS: 149092-35-3)

AG 555 (Calbiochem Cat. No. 658404) (CAS: 133550-34-2)

AG 494 (Tocris Cat. No. 0619) (CAS: 133550-35-3)

PD168393 (Calbiochem Cat. No. 513033)

Tyrphostin B44 (+ and − isomers) (Calbiochem Cat. No. 658402) (CAS:133550-32-0)

AG 490 Tyrphostin B42 (Calbiochem. Cat. No. 658401) (CAS: 133550-30-8)

PI3K LY294002 (Calbiochem. Cat. No. 440202) (CAS: 154447-36-6)

Cdk5 Seliciclib (R- roscovitine/CYC202) (CAS: 186692-46-6)

Adenylyl cyclase Forskolin (Calbiochem Cat. No. 344270) (CAS:66575-29-9)

NKH 477 (Tocris Cat. No. 1603) (CAS: 138605-00-2)

Transcription Actinomycin D (Calbiochem Cat. No. 114666) (CAS: 50-76-0)

JNK SP600125 (Calbiochem Cat. No. 420119) (CAS: 129-56-6)

JNKV Inhibitor AS601245 (Calbiochem Cat. No. 420129

JNKVIII Inhibitor Calbiochem Cat. No. 420135

Bax Channel Bax Channel Blocker (Tocris Cat. No. 2160) (CAS:335165-68-9)

Ih Channel ZD7288 (Tocris Cat. No. 1000) (CAS: 133059-99-1)

CAMK STO-609 (Calbiochem Cat. No. 570250) (CAS: 52029-86-4)

Protein Synthesis Anisomycin (CAS: 22862-76-6)

Cycloheximide (CAS: 66-81-9)

TABLE 3 Target Compounds Structure CAS/Reference Proteasome Bortezomib[(1R)-3-methyl-1-({(2S)-3- CAS: 179324- (Velcade ™)phenyl-2-[(pyrazin-2- 69-7 ylcarbonyl)amino]propanoyl} Adams et al.,amino)butyl]boronic acid Cancer Invest. 22 (2): 304 (2004) Disulfiram 1-CAS: 97-77-8 (Antabus ™/ (diethylthiocarbamoyldisulfanyl)- Lövborg etal., Antabuse ™) N,N-diethyl- International methanethioamide Journal ofCancer 118 (6): 1577 (2006) GSK3β Lithium Salts CAS: 7447-41-8 (e.g.,lithium Kaladchibachi et carbonate (e.g., al., J. Circadian Lithobid ™)and Rhythms 5: 3 lithium citrate) (2007) p38 MAPK PamapimodPyrido[2,3-d]pyrimidin-7(8H)- CAS: 449811- one, 6-(2,4-difluorophenoxy)-01-2 2-[[3-hydroxy-1-(2- Hill et al., J. hydroxyethyl)propyl]amino]-8-Pharmacol Exp. methyl- Therapeutics Online publication (September 2008)EGFRK Gefitinib N-(3-chloro-4-fluoro-phenyl)- CAS: 184475-35-2(Iressa ™) 7-methoxy- Paez et al., 6-(3-morpholin-4- Scienceylpropoxy)quinazolin-4-amine 304(5676): 1497 (2004) ErlotinibN-(3-ethynylphenyl)-6,7-bis(2- CAS: 183321- (Tarceva ™) methoxyethoxy)74-6 quinazolin-4-amine Shepherd et al., N. Engl. J. Med. 353: 123(2005) Lapatinib N-[3-chloro-4-[(3- CAS: 388082- ditosylatefluorophenyl)methoxy]phenyl]- 78-8 (Tykerb ™/ 6-[5-[(2- Higa et al.,Exp. Tyverb ™) methylsulfonylethylamino)methyl]- Rev. Anticancer2-furyl]quinazolin-4-amine Ther. 7(9): 1183 (2007) AdenylylDemeclocycline 2-(amino-hydroxy- CAS: 127-33-3 cyclase hydrochloridemethylidene)-7-chloro-4- Roitman et al., (Declomycin ™/dimethylamino-6,10,11,12a- Human Declostatin ™/tetrahydroxy-4,4a,5,5a,6,12a- Psychopharma- Ledermycin ™)hexahydrotetracene-1,3,12- cololgy: Clinical trione and Experimental13(2): 121-125 (1998) Protein Gentamicin 2-[4,6-diamino-3-[3-amino-6-CAS: 1405-41-0 Synthesis sulfate (1-methylaminoethyl) Kadurugamuwa(Garamycin ™) tetrahydropyran-2-yl]oxy-2- et al., J.hydroxy-cyclohexoxy]-5- Bacteriol. methyl-4-methylamino- 175: 5798(1993) tetrahydropyran-3,5-diol Neomycin (1R,2R,3S,4R,6S)-4,6- CAS:1404-04-2 sulfate diamino-2-{[3-O-(2,6- Hu, Proc. Natl. (Mycifradin ™,diamino-2,6-dideoxy-β-L- Acad. Sci. USA Neo-Rx ™) idopyranosyl)-β-D-95(17): 9791 ribofuranosyl]oxy}-3- (1998) hydroxycyclohexyl 2,6-diamino-2,6-dideoxy-α-D- glucopyranoside Paromomycin(2R,3S,4R,5R,6S)-5-amino-6- CAS: 1263-89-4 sulfate [(1R,2S,3S,4R,6S)-VanLoock et al., (Humatin ™) 4,6-diamino-2- J. Molecular[(2S,3R,4R,5R)-4- Biol. 304(4): 507 [(2R,3R,4R,5R,6S)- (2000)3-amino-6-(aminomethyl)-4,5- dihydroxy-oxan-2-yl] oxy-3-hydroxy-5-(hydroxymethyl)oxolan-2- yl]oxy- 3-hydroxy-cyclohexyl]oxy-2-(hydroxymethyl)oxane-3,4-diol

D. Making Antibody Inhibitors of Neuron or Axon Degeneration

Antibodies identified by the binding and activity assays of the presentinvention can be produced by methods known in the art, includingtechniques of recombinant DNA technology.

(i) Antigen Preparation

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g.,the extracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.,cancer cell lines) or may be cells that have been transformed byrecombinant techniques to express the transmembrane molecule. Exemplarysequences of targets of the invention are referred to in Table 1, andcan be used in the preparation of antigens for making antibodies for usein the invention. Other antigens and forms thereof useful for preparingantibodies will be apparent to those in the art.

(ii) Polyclonal Antibodies

Polyclonal antibodies are typically raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCI₂, or R₁N═C═NR, whereR and R₁ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 of theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and serum is assayed for antibody titer. Animals areboosted until the titer plateaus. The animal can be boosted with aconjugate of the same antigen, but conjugated to a different proteinand/or through a different cross-linking reagent. Conjugates also can bemade in recombinant cell culture as protein fusions. Also, aggregatingagents such as alum are suitably used to enhance the immune response.

(iii) Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature 256:495, 1975, or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). In thehybridoma method, a mouse or other appropriate host animal, such as ahamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103, Academic Press, 1986).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that can contain one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. For example,if the parental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Exemplary myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,particular myeloma cell lines that may be considered for use are murinemyeloma lines, such as those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif., USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Manassas, Va., USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001,1984; Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63, Marcel Dekker, Inc., New York, 1987).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen. Thebinding specificity of monoclonal antibodies produced by hybridoma cellscan be determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbentassay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, clones may be subclonedby limiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103, AcademicPress, 1986). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Recombinant production of antibodies will be described in moredetail below.

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed, for example, in McCafferty et al., Nature 348:552-554, 1990.

Clackson et al., Nature 352:624-628, 1991 and Marks et al., J. Mol.Biol. 222:581-597, 1991, describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology 10:779-783, 1992), as wellas combinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nucl. Acids.Res. 21:2265-2266, 1993). Thus, these techniques are viable alternativesto traditional monoclonal antibody hybridoma techniques for isolation ofmonoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal., Proc. Natl. Acad. Sci. U.S.A. 81:6851, 1984), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically, such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iv) Humanized and Human Antibodies

A humanized antibody has one or more amino acid residues introduced intoit from a source that is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature 321:522-525, 1986; Riechmann et al., Nature 332:323-327, 1988;Verhoeyen et al., Science 239:1534-1536, 1988), by substituting rodentCDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit”method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence that is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol. 151:2296, 1993; Chothia et al., J. Mol. Biol. 196:901, 1987).Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. U.S.A.89:4285, 1992; Presta et al., J. Immnol. 151:2623, 1993).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to an exemplary method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablethat illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. U.S.A. 90:2551,1993; Jakobovits et al., Nature 362:255-258, 1993; Bruggermann et al.,Year in Immunol. 7:33, 1993; and Duchosal et al., Nature 355:258, 1992.Human antibodies can also be derived from phage-display libraries(Hoogenboom et al., J. Mol. Biol. 227:381, 1991; Marks et al., J. Mol.Biol. 222:581-597, 1991; Vaughan et al., Nature Biotech. 14:309, 1996).Generation of human antibodies from antibody phage display libraries isfurther described below.

(v) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem.Biophys. Meth. 24:107-117, 1992 and Brennan et al., Science 229:81,1985). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167, 1992). In another embodiment as described inthe example below, the F(ab′)₂ is formed using the leucine zipper GCN4to promote assembly of the F(ab′)₂ molecule. According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner. In otherembodiments, the antibody of choice is a single chain Fv fragment (scFv)(see WO 93/16185).

(vi) Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent epitopes, where the epitopes are usually from differentantigens. While such molecules normally will only bind two differentepitopes (i.e., bispecific antibodies, BsAbs), antibodies withadditional specificities such as trispecific antibodies are encompassedby this expression when used herein.

Methods for making bispecific antibodies are known in the art.Traditional production of full-length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539, 1983). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ. 10:3655-3659, 1991. According to a different approach, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion can be with an immunoglobulin heavy chainconstant domain, comprising at least part of the hinge, CH2, and CH3regions. The first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding can be present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In one example of this approach, the bispecific antibodies are composedof a hybrid immunoglobulin heavy chain with a first binding specificityin one arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. It was foundthat this asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. Thisapproach is disclosed in WO 94/04690. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods Enzymol.121:210, 1986.

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers that are recovered from recombinant cellculture. The interface may comprise at least a part of the CH3 domain ofan antibody constant domain. In this method, one or more small aminoacid side chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360 and WO 92/200373). Heteroconjugate antibodies may be made usingany convenient cross-linking methods. Suitable cross-linking agents arewell known in the art and are disclosed in U.S. Pat. No. 4,676,980,along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81, 1985 describe a procedure in which intact antibodies areproteolytically cleaved to generate F(ab′)2 fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′-SH fragments can also be directly recovered from E. coli, and canbe chemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225, 1992 describe the production of a fully humanizedbispecific antibody F(ab′)2 molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers (Kostelny et al., J. Immunol. 148(5):1547-1553, 1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. U.S.A. 90:6444-6448, 1993, has provided an alternative mechanismfor making bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker that is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported (see Gruber et al.,J. Immunol. 152:5368, 1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared (Tuft et al., J. Immunol. 147:60,1991).

(vii) Effector Function Engineering

It may be desirable to modify an antibody used in the invention withrespect to effector function, so as to enhance the effectiveness of theantibody. For example cysteine residue(s) may be introduced in the Fcregion, thereby allowing interchain disulfide bond formation in thisregion. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC; see Caron etal., J. Exp. Med. 176:1191-1195, 1992 and Shopes, J. Immunol.148:2918-2922, 1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research 53:2560-2565, 1993.Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities(see Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

(viii) Antibody-Salvage Receptor Binding Epitope Fusions

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase bloodbrain barrier penetration, for example. In this case, it may bedesirable to modify the antibody fragment in order to increase its serumhalf-life. This may be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis).

The salvage receptor binding epitope can constitute a region in whichany one or more amino acid residues from one or two loops of an Fcdomain are transferred to an analogous position of the antibodyfragment. In another example, three or more residues from one or twoloops of the Fc domain are transferred, while in a further example, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or V_(H) region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the CL region or VLregion, or both, of the antibody fragment.

(ix) Other Covalent Modifications of Antibodies

Covalent modifications of antibodies are included within the scope ofthis invention. They may be made by chemical synthesis or by enzymaticor chemical cleavage of the antibody, if applicable. Other types ofcovalent modifications of the antibody are introduced into the moleculeby reacting targeted amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues. Examples of covalent modifications aredescribed in U.S. Pat. No. 5,534,615, which is specifically incorporatedherein by reference. One example of a type of covalent modification ofthe antibody comprises linking the antibody to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.

(x) Generation of Antibodies from Synthetic Antibody Phage Libraries

In a further embodiment, the invention may employ a method forgenerating and selecting novel antibodies using a phage displayapproach. The approach involves generation of synthetic antibody phagelibraries based on single framework template, design of sufficientdiversities within variable domains, display of polypeptides having thediversified variable domains, selection of candidate antibodies withhigh affinity to target the antigen, and isolation of the selectedantibodies.

Details of phage display methods can be found, for example, WO03/102157, the entire disclosure of which is expressly incorporatedherein by reference.

In one example, the antibody libraries used in the invention can begenerated by mutating the solvent accessible and/or highly diversepositions in at least one CDR of an antibody variable domain. Some orall of the CDRs can be mutated using the methods provided herein. Insome embodiments, diverse antibody libraries can be generated bymutating positions in CDRH1, CDRH2, and CDRH3 to form a single library,or by mutating positions in CDRL3 and CDRH3 to form a single library, orby mutating positions in CDRL3 and CDRH1, CDRH2, and CDRH3 to form asingle library.

A library of antibody variable domains can be generated, for example,having mutations in the solvent accessible and/or highly diversepositions of CDRH1, CDRH2, and CDRH3. Another library can be generatedhaving mutations in CDRL1, CDRL2, and CDRL3. These libraries can also beused in conjunction with each other to generate binders of desiredaffinities. For example, after one or more rounds of selection of heavychain libraries for binding to a target antigen, a light chain librarycan be replaced into the population of heavy chain binders for furtherrounds of selection to increase the affinity of the binders.

A library can be created by substitution of original amino acids withvariant amino acids in the CDRH3 region of the variable region of theheavy chain sequence. The resulting library can contain a plurality ofantibody sequences, in which the sequence diversity is primarily in theCDRH3 region of the heavy chain sequence.

In one example, the library is created in the context of the humanizedantibody 4D5 sequence, or the sequence of the framework amino acids ofthe humanized antibody 4D5 sequence. The library can be created bysubstitution of at least residues 95-100a of the heavy chain with aminoacids encoded by the DVK codon set, wherein the DVK codon set is used toencode a set of variant amino acids for every one of these positions. Anexample of an oligonucleotide set that is useful for creating thesesubstitutions comprises the sequence (DVK)₇. In some embodiments, alibrary is created by substitution of residues 95-100a with amino acidsencoded by both DVK and NNK codon sets. An example of an oligonucleotideset that is useful for creating these substitutions comprises thesequence (DVK)₆ (NNK). In another embodiment, a library is created bysubstitution of at least residues 95-100a with amino acids encoded byboth DVK and NNK codon sets. An example of an oligonucleotide set thatis useful for creating these substitutions comprises the sequence (DVK)₅(NNK). Another example of an oligonucleotide set that is useful forcreating these substitutions comprises the sequence (NNK)₆. Otherexamples of suitable oligonucleotide sequences can be determined by oneskilled in the art according to the criteria described herein.

In another embodiment, different CDRH3 designs are utilized to isolatehigh affinity binders and to isolate binders for a variety of epitopes.The range of lengths of CDRH3 generated in this library is 11 to 13amino acids, although lengths different from this can also be generated.H3 diversity can be expanded by using NNK, DVK, and NVK codon sets, aswell as more limited diversity at N and/or C-terminal.

Diversity can also be generated in CDRH1 and CDRH2. The designs ofCDR-H1 and H2 diversities follow the strategy of targeting to mimicnatural antibodies repertoire as described with modification that focusthe diversity more closely matched to the natural diversity thanprevious design.

For diversity in CDRH3, multiple libraries can be constructed separatelywith different lengths of H3 and then combined to select for binders totarget antigens. The multiple libraries can be pooled and sorted usingsolid support selection and solution-sorting methods as describedpreviously and herein below. Multiple sorting strategies may beemployed. For example, one variation involves sorting on target bound toa solid, followed by sorting for a tag that may be present on the fusionpolypeptide (e.g., anti-gD tag) and followed by another sort on targetbound to solid. Alternatively, the libraries can be sorted first ontarget bound to a solid surface, the eluted binders are then sortedusing solution phase binding with decreasing concentrations of targetantigen. Utilizing combinations of different sorting methods providesfor minimization of selection of only highly expressed sequences andprovides for selection of a number of different high affinity clones.

High affinity binders for the target antigen can be isolated from thelibraries. Limiting diversity in the H1/H2 region decreases degeneracyabout 10⁴ to 10⁵ fold and allowing more H3 diversity provides for morehigh affinity binders. Utilizing libraries with different types ofdiversity in CDRH3 (e.g., utilizing DVK or NVT) provides for isolationof binders that may bind to different epitopes of a target antigen.

Of the binders isolated from the pooled libraries as described above, ithas been discovered that affinity may be further improved by providinglimited diversity in the light chain. Light chain diversity is generatedin this embodiment as follows in CDRL1: amino acid position 28 isencoded by RDT; amino acid position 29 is encoded by RKT; amino acidposition 30 is encoded by RVW; amino acid position 31 is encoded by ANW;amino acid position 32 is encoded by THT; optionally, amino acidposition 33 is encoded by CTG; in CDRL2: amino acid position 50 isencoded by KBG; amino acid position 53 is encoded by AVC; andoptionally, amino acid position 55 is encoded by GMA; in CDRL3: aminoacid position 91 is encoded by TMT or SRT or both; amino acid position92 is encoded by DMC; amino acid position 93 is encoded by RVT; aminoacid position 94 is encoded by NHT; and amino acid position 96 isencoded by TWT or YKG or both.

In another embodiment, a library or libraries with diversity in CDRH1,CDRH2, and CDRH3 regions is generated. In this embodiment, diversity inCDRH3 is generated using a variety of lengths of H3 regions and usingprimarily codon sets XYZ and NNK or NNS. Libraries can be formed usingindividual oligonucleotides and pooled or oligonucleotides can be pooledto form a subset of libraries. The libraries of this embodiment can besorted against target bound to solid. Clones isolated from multiplesorts can be screened for specificity and affinity using ELISA assays.For specificity, the clones can be screened against the desired targetantigens as well as other nontarget antigens. Those binders to thetarget antigen can then be screened for affinity in solution bindingcompetition ELISA assay or spot competition assay. High affinity binderscan be isolated from the library utilizing XYZ codon sets prepared asdescribed above. These binders can be readily produced as antibodies orantigen binding fragments in high yield in cell culture.

In some embodiments, it may be desirable to generate libraries with agreater diversity in lengths of CDRH3 region. For example, it may bedesirable to generate libraries with CDRH3 regions ranging from about 7to 19 amino acids.

High affinity binders isolated from the libraries of these embodimentsare readily produced in bacterial and eukaryotic cell culture in highyield. The vectors can be designed to readily remove sequences such asgD tags, viral coat protein component sequence, and/or to add inconstant region sequences to provide for production of full lengthantibodies or antigen binding fragments in high yield.

A library with mutations in CDRH3 can be combined with a librarycontaining variant versions of other CDRs, for example CDRL1, CDRL2,CDRL3, CDRH1, and/or CDRH2. Thus, for example, in one embodiment, aCDRH3 library is combined with a CDRL3 library created in the context ofthe humanized 4D5 antibody sequence with variant amino acids atpositions 28, 29, 30, 31, and/or 32 using predetermined codon sets. Inanother embodiment, a library with mutations to the CDRH3 can becombined with a library comprising variant CDRH1 and/or CDRH2 heavychain variable domains. In one embodiment, the CDRH1 library is createdwith the humanized antibody 4D5 sequence with variant amino acids atpositions 28, 30, 31, 32, and 33. A CDRH2 library may be created withthe sequence of humanized antibody 4D5 with variant amino acids atpositions 50, 52, 53, 54, 56, and 58 using the predetermined codon sets.

(xi) Antibody Mutants

The antibodies generated from phage libraries can be further modified togenerate antibody mutants with improved physical, chemical, and/orbiological properties over the parent antibody. Where the assay used isa biological activity assay, the antibody mutant can have a biologicalactivity in the assay of choice that is at least about 10 fold better,at least about 20 fold better, at least about 50 fold better, andsometimes at least about 100 fold or 200 fold better, than thebiological activity of the parent antibody in that assay. For example,an anti-target antibody mutant may have a binding affinity for thetarget that is at least about 10 fold stronger, at least about 20 foldstronger, at least about 50 fold stronger, and sometimes at least about100 fold or 200 fold stronger, than the binding affinity of the parentantibody.

To generate the antibody mutant, one or more amino acid alterations(e.g., substitutions) are introduced in one or more of the hypervariableregions of the parent antibody. Alternatively, or in addition, one ormore alterations (e.g., substitutions) of framework region residues maybe introduced in the parent antibody where these result in animprovement in the binding affinity of the antibody mutant for theantigen from the second mammalian species. Examples of framework regionresidues to modify include those that non-covalently bind antigendirectly (Amit et al., Science 233:747-753, 1986); interact with/affectthe conformation of a CDR (Chothia et al., J. Mol. Biol. 196:901-917,1987); and/or participate in the V_(L)-V_(H) interface (EP 239 400B1).In certain embodiments, modification of one or more of such frameworkregion residues results in an enhancement of the binding affinity of theantibody for the antigen from the second mammalian species. For example,from about one to about five framework residues may be altered in thisembodiment of the invention. Sometimes, this may be sufficient to yieldan antibody mutant suitable for use in preclinical trials, even wherenone of the hypervariable region residues have been

altered. Normally, however, the antibody mutant will comprise additionalhypervariable region alteration(s).

The hypervariable region residues that are altered may be changedrandomly, especially where the starting binding affinity of the parentantibody is such that such randomly produced antibody mutants can bereadily screened.

One useful procedure for generating such antibody mutants is called“alanine scanning mutagenesis” (Cunningham et al., Science244:1081-1085, 1989). Here, one or more of the hypervariable regionresidue(s) are replaced by alanine or polyalanine residue(s) to affectthe interaction of the amino acids with the antigen from the secondmammalian species. Those hypervariable region residue(s) demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other mutations at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. The ala-mutants produced this way arescreened for their biological activity as described herein.

Normally one would start with a conservative substitution such as thoseshown below under the heading of “preferred substitutions.” If suchsubstitutions result in a change in biological activity (e.g., bindingaffinity), then more substantial changes, denominated “exemplarysubstitutions” in the Table 4, or as further described below inreference to amino acid classes, are introduced and the productsscreened.

TABLE 4 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; met;ala; ile phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F)leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) serser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; ala; leu norleucine

Even more substantial modifications in the antibodies biologicalproperties are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr, asn, gln;

(3) acidic: asp, glu;

(4) basic: his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

In another embodiment, the sites selected for modification are affinitymatured using phage display (see above).

Nucleic acid molecules encoding amino acid sequence mutants are preparedby a variety of methods known in the art. These methods include, but arenot limited to, oligonucleotide-mediated (or site-directed) mutagenesis(see, e.g., Kunkel, Proc. Natl. Acad. Sci. USA 82:488 (1985)), PCRmutagenesis, and cassette mutagenesis of an earlier prepared mutant or anon-mutant version of the parent antibody.

In certain embodiments, the antibody mutant will only have a singlehypervariable region residue substituted. In other embodiments, two ormore of the hypervariable region residues of the parent antibody willhave been substituted, e.g., from about two to about ten hypervariableregion substitutions.

Ordinarily, the antibody mutant with improved biological properties willhave an amino acid sequence having at least 75% amino acid sequenceidentity or similarity with the amino acid sequence of either the heavyor light chain variable domain of the parent antibody, for example, atleast 80%, at least 85%, at least 90%, or at least 95% sequence identityor similarity. Identity or similarity with respect to this sequence isdefined herein as the percentage of amino acid residues in the candidatesequence that are identical (i.e., same residue) or similar (i.e., aminoacid residue from the same group based on common side-chain properties,see above) with the parent antibody residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence outsideof the variable domain shall be construed as affecting sequence identityor similarity.

Following production of the antibody mutant, the biological activity ofthat molecule relative to the parent antibody is determined. As notedabove, this may involve determining the binding affinity and/or otherbiological activities of the antibody. In a preferred embodiment of theinvention, a panel of antibody mutants is prepared and screened forbinding affinity for the antigen or a fragment thereof. One or more ofthe antibody mutants selected from this initial screen are optionallysubjected to one or more further biological activity assays to confirmthat the antibody mutant(s) with enhanced binding affinity are indeeduseful, e.g., for preclinical studies.

The antibody mutant(s) so selected may be subjected to furthermodifications, oftentimes depending on the intended use of the antibody.Such modifications may involve further alteration of the amino acidsequence, fusion to heterologous polypeptide(s) and/or covalentmodifications such as those elaborated below. With respect to amino acidsequence alterations, exemplary modifications are elaborated above. Forexample, any cysteine residue not involved in maintaining the properconformation of the antibody mutant also may be substituted, generallywith serine, to improve the oxidative stability of the molecule andprevent aberrant cross linking. Conversely, cysteine bond(s) may beadded to the antibody to improve its stability (particularly where theantibody is an antibody fragment such as an Fv fragment). Another typeof amino acid mutant has an altered glycosylation pattern. This may beachieved by deleting one or more carbohydrate moieties found in theantibody, and/or adding one or more glycosylation sites that are notpresent in the antibody. Glycosylation of antibodies is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to theantibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

(xii) Recombinant Production of Antibodies

For recombinant production of an antibody, a nucleic acid encoding theantibody is isolated and inserted into a replicable vector for furthercloning (amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence(e.g., as described in U.S. Pat. No. 5,534,615, which is specificallyincorporated herein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serrafia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710), Pseudomonas such as P. aeruginosa, andStreptomyces. One exemplary E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X 1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subloned for growth insuspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. U.S.A. 77:4216,1980); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-25, 1980);monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68,1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce antibodies for use in the invention maybe cultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44, 1979, Barnes et al., Anal.Biochem. 102:255, 1980, U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™), trace elements (defined as inorganiccompounds usually present at final concentrations in the micromolarrange), and glucose or an equivalent energy source. Any other necessarysupplements may also be included at appropriate concentrations thatwould be known to those skilled in the art. The culture conditions, suchas temperature, pH, and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed cells, is removed,for example, by centrifugation or ultrafiltration. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13, 1983). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:1567-1575, 1986). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipiation are alsoavailable depending on the antibody to be recovered.

E. Use of Inhibitors of Neuron or Axon Degeneration

Inhibitors of the targets described herein, such as inhibitorsidentified or characterized in the screening assays described herein(e.g., the particular inhibitors described above) can be used in methodsfor inhibiting neuron or axon degeneration. The inhibitors are,therefore, useful in the therapy of, for example, (i) disorders of thenervous system (e.g., neurodegenerative diseases), (ii) conditions ofthe nervous system that are secondary to a disease, condition, ortherapy having a primary effect outside of the nervous system, (iii)injuries to the nervous system caused by physical, mechanical, orchemical trauma, (iv) pain, (v) ocular-related neurodegeneration, (vi)memory loss, and (vii) psychiatric disorders. Non-limiting examples ofsome of these diseases, conditions, and injuries are provided below.

Examples of neurodegenerative diseases and conditions that can beprevented or treated according to the invention include amyotrophiclateral sclerosis (ALS), trigeminal neuralgia, glossopharyngealneuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy,progressive muscular atrophy, primary lateral sclerosis (PLS),pseudobulbar palsy, progressive bulbar palsy, spinal muscular atrophy,progressive bulbar palsy, inherited muscular atrophy, invertebrate disksyndromes (e.g., herniated, ruptured, and prolapsed disk syndromes),cervical spondylosis, plexus disorders, thoracic outlet destructionsyndromes, peripheral neuropathies, prophyria, mild cognitiveimpairment, Alzheimer's disease, Huntington's disease, Parkinson'sdisease, Parkinson's-plus diseases (e.g., multiple system atrophy,progressive supranuclear palsy, and corticobasal degeneration), dementiawith Lewy bodies, frontotemporal dementia, demyelinating diseases (e.g.,Guillain-Barré syndrome and multiple sclerosis), Charcot-Marie-Toothdisease (CMT; also known as Hereditary Motor and Sensory Neuropathy(HMSN), Hereditary Sensorimotor Neuropathy (HSMN), and Peroneal MuscularAtrophy), prion disease (e.g., Creutzfeldt-Jakob disease,Gerstmann-Sträussler-Scheinker syndrome (GSS), fatal familial insomnia(FFI), and bovine spongiform encephalopathy (BSE, commonly known as madcow disease)), Pick's disease, epilepsy, and AIDS demential complex(also known as HIV dementia, HIV encephalopathy, and HIV-associateddementia).

The methods of the invention can also be used in the prevention andtreatment of ocular-related neurodegeneration and related diseases andconditions, such as glaucoma, lattice dystrophy, retinitis pigmentosa,age-related macular degeneration (AMD), photoreceptor degenerationassociated with wet or dry AMD, other retinal degeneration, optic nervedrusen, optic neuropathy, and optic neuritis. Non-limiting examples ofdifferent types of glaucoma that can be prevented or treated accordingto the invention include primary glaucoma (also known as primaryopen-angle glaucoma, chronic open-angle glaucoma, chronic simpleglaucoma, and glaucoma simplex), low-tension glaucoma, primaryangle-closure glaucoma (also known as primary closed-angle glaucoma,narrow-angle glaucoma, pupil-block glaucoma, and acute congestiveglaucoma), acute angle-closure glaucoma, chronic angle-closure glaucoma,intermittent angle-closure glaucoma, chronic open-angle closureglaucoma, pigmentary glaucoma, exfoliation glaucoma (also known aspseudoexfoliative glaucoma or glaucoma capsulare), developmentalglaucoma (e.g., primary congenital glaucoma and infantile glaucoma),secondary glaucoma (e.g., inflammatory glaucoma (e.g., uveitis and Fuchsheterochromic iridocyclitis)), phacogenic glaucoma (e.g., angle-closureglaucoma with mature cataract, phacoanaphylactic glaucoma secondary torupture of lens capsule, phacolytic glaucoma due to phacotoxic meshworkblockage, and subluxation of lens), glaucoma secondary to intraocularhemorrhage (e.g., hyphema and hemolytic glaucoma, also known aserythroclastic glaucoma), traumatic glaucoma (e.g., angle recessionglaucoma, traumatic recession on anterior chamber angle, postsurgicalglaucoma, aphakic pupillary block, and ciliary block glaucoma),neovascular glaucoma, drug-induced glaucoma (e.g., corticosteroidinduced glaucoma and alpha-chymotrypsin glaucoma), toxic glaucoma, andglaucoma

associated with intraocular tumors, retinal deatchments, severe chemicalburns of the eye, and iris atrophy.

Examples of types of pain that can be treated according to the methodsof the invention include those associated with the following conditions:chronic pain, fibromyalgia, spinal pain, carpel tunnel syndrome, painfrom cancer, arthritis, sciatica, headaches, pain from surgery, musclespasms, back pain, visceral pain, pain from injury, dental pain,neuralgia, such as neuogenic or neuropathic pain, nerve inflammation ordamage, shingles, herniated disc, torn ligament, and diabetes.

Certain diseases and conditions having primary effects outside of thenervous system can lead to damage to the nervous system, which can betreated according to the methods of the present invention. Examples ofsuch conditions include peripheral neuropathy and neuralgia caused by,for example, diabetes, cancer, AIDS, hepatitis, kidney dysfunction,Colorado tick fever, diphtheria, HIV infection, leprosy, lyme disease,polyarteritis nodosa, rheumatoid arthritis, sarcoidosis, Sjogrensyndrome, syphilis, systemic lupus erythematosus, and amyloidosis.

In addition, the methods of the invention can be used in the treatmentof nerve damage, such as peripheral neuropathy, which is caused byexposure to toxic compounds, including heavy metals (e.g., lead,arsenic, and mercury) and industrial solvents, as well as drugsincluding chemotherapeutic agents (e.g., vincristine and cisplatin),dapsone, HIV medications (e.g., Zidovudine, Didanosine, Stavudine,Zalcitabine, Ritonavir, and Amprenavir), cholesterol lowering drugs(e.g., Lovastatin, Indapamid, and Gemfibrozil), heart or blood pressuremedications (e.g., Amiodarone, Hydralazine, Perhexiline), andMetronidazole.

The methods of the invention can also be used to treat injury to thenervous system caused by physical, mechanical, or chemical trauma. Thus,the methods can be used in the treatment of peripheral nerve damagecaused by physical injury (associated with, e.g., burns, wounds,surgery, and accidents), ischemia, prolonged exposure to coldtemperature (e.g., frost-bite), as well as damage to the central nervoussystem due to, e.g., stroke or intracranial hemorrhage (such as cerebralhemorrhage).

Further, the methods of the invention can be used in the prevention ortreatment of memory loss such as, for example, age-related memory loss.Types of memory that can be affected by loss, and thus treated accordingto the invention, include episodic memory, semantic memory, short-termmemory, and long-term memory. Examples of diseases and conditionsassociated with memory loss, which can be treated according to thepresent invention, include mild cognitive impairment, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, chemotherapy,stress, stroke, and traumatic brain injury (e.g., concussion).

The methods of the invention can also be used in the treatment ofpsychiatric disorders including, for example, schizophrenia, delusionaldisorder, schizoaffective disorder, schizopheniform, shared psychoticdisorder, psychosis, paranoid personality disorder, schizoid personalitydisorder, borderline personality disorder, anti-social personalitydisorder, narcissistic personality disorder, obsessive-compulsivedisorder, delirium, dementia, mood disorders, bipolar disorder,depression, stress disorder, panic disorder, agoraphobia, social phobia,post-traumatic stress disorder, anxiety disorder, and impulse controldisorders (e.g., kleptomania, pathological gambling, pyromania, andtrichotillomania).

In addition to the in vivo methods described above, the methods of theinvention can be used to treat nerves ex vivo, which may be helpful inthe context of nerve grafts or nerve transplants. Thus, the inhibitorsdescribed herein can be useful as components of culture media for use inculturing nerve cells in vitro.

Therapeutic formulations of the inhibitors described herein are preparedfor storage by mixing the inhibitor (such as small molecule or anantibody) having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers (see,e.g., Remington's Pharmaceutical Sciences (18^(th) edition), ed. A.Gennaro, 1990, Mack Publishing Co., Easton, Pa.), in the form oflyophilized cake or aqueous solutions. Acceptable carriers, excipients,or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and can include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid,BHA, and BHT; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counter-ions such assodium; and/or nonionic surfactants such as Tween, Pluronics, or PEG.

Inhibitors to be used for in vivo administration must be sterile, whichcan be achieved by filtration through sterile filtration membranes,prior to or following lyophilization and reconstitution. Therapeuticcompositions may be placed into a container having a sterile accessport, for example, an intravenous solution bag or vial having a stopperpierceable by a hypodermic injection needle.

The inhibitors can be optionally combined with or administered inconcert with each other or other agents known to be useful in thetreatment of the relevant disease or condition. Thus, in the treatmentof ALS, for example, inhibitors can be administered in combination withRiluzole (Rilutek), minocycline, insulin-like growth factor 1 (IGF-1),and/or methylcobalamin. In another example, in the treatment ofParkinson's disease, inhibitors can be administered with L-dopa,dopamine agonists (e.g., bromocriptine, pergolide, pramipexole,ropinirole, cabergoline, apomorphine, and lisuride), dopa decarboxylaseinhibitors (e.g., levodopa, benserazide, and carbidopa), and/or MAO-Binhibitors (e.g., selegiline and rasagiline). In a further example, inthe treatment of Alzheimer's disease, inhibitors can be administeredwith acetylcholinesterase inhibitors (e.g., donepezil, galantamine, andrivastigmine) and/or NMDA receptor antagonists (e.g., memantine). Thecombination therapies can involve concurrent or sequentialadministration, by the same or different routes, as determined to beappropriate by those of skill in the art. The invention also includespharmaceutical compositions and kits including combinations as describedherein.

In addition to the combinations noted above, other combinations includedin the invention are combinations of inhibitors of degeneration ofdifferent neuronal regions. Thus, the invention includes combinations ofagents that (i) inhibit degeneration of the neuron cell body, and (ii)inhibit axon degeneration. As described further below, inhibitors of GSKand transcription were found to prevent degeneration of neuron cellbodies, while inhibitors of EGFR and p38 MAPK prevent degeneration ofaxons. Thus, the invention includes combinations of inhibitors of GSKand EGFR (and/or p38 MAPK), combinations of transcription inhibitors andEGFR (and/or p38 MAPK), and further combinations of inhibitors of dualleucine zipper-bearing kinase (DLK), glycogen synthase kinase 3β(GSK3β), p38 MAPK, EGFF, phosphoinositide 3-kinase (PI3K),cyclin-dependent kinase 5 (cdk5), adenylyl cyclase, c-Jun N-terminalkinase (JNK), BCL2-associated X protein (Bax), In channel,calcium/calmodulin-dependent protein kinase kinase (CaMKK), a G-protein,a G-protein coupled receptor, transcription factor 4 (TCF4), andβ-catenin. The inhibitors used in these combinations can be any of thosedescribed herein, or other inhibitors of these targets.

The route of administration of the inhibitors is selected in accordancewith known methods, e.g., injection or infusion by intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial or intralesional routes, topical administration, or bysustained release systems as described below.

For intracerebral use, the compounds can be administered continuously byinfusion into the fluid reservoirs of the CNS, although bolus injectionmay be acceptable. The inhibitors can be administered into theventricles of the brain or otherwise introduced into the CNS or spinalfluid. Administration can be performed by use of an indwelling catheterand a continuous administration means such as a pump, or it can beadministered by implantation, e.g., intracerebral implantation of asustained-release vehicle. More specifically, the inhibitors can beinjected through chronically implanted cannulas or chronically infusedwith the help of osmotic minipumps. Subcutaneous pumps are availablethat deliver proteins through a small tubing to the cerebral ventricles.Highly sophisticated pumps can be refilled through the skin and theirdelivery rate can be set without surgical intervention. Examples ofsuitable administration protocols and delivery systems involving asubcutaneous pump device or continuous intracerebroventricular infusionthrough a totally implanted drug delivery system are those used for theadministration of dopamine, dopamine agonists, and cholinergic agoniststo Alzheimer's disease patients and animal models for Parkinson'sdisease, as described by Harbaugh, J. Neural Transm. Suppl. 24:271,1987; and DeYebenes et al., Mov. Disord. 2:143, 1987.

Suitable examples of sustained release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.,films or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers 22:547, 1983), poly(2-hydroxyethyl-methacrylate) (Langer etal., J. Biomed. Mater. Res. 15:167, 1981; Langer, Chem. Tech. 12:98,1982), ethylene vinyl acetate (Langer, et al., Id), orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988A). Sustained releasecompositions also include liposomally entrapped compounds, which can beprepared by methods known per se (Epstein et al., Proc. Natl. Acad. Sci.U.S.A. 82:3688, 1985; Hwang et al., Proc. Natl. Acad. Sci. U.S.A.77:4030, 1980; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324A).Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamelar type in which the lipid content is greater than about 30 mol% cholesterol, the selected proportion being adjusted for the optimaltherapy.

An effective amount of an active compound to be employed therapeuticallywill depend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.A typical daily dosage might range from, for example, about 1 μg/kg toup to 100 mg/kg or more (e.g., about 1 μg/kg to 1 mg/kg, about 1 μg/kgto about 5 mg/kg, about 1 mg/kg to 10 mg/kg, about 5 mg/kg to about 200mg/kg, about 50 mg/kg to about 150 mg/mg, about 100 mg/kg to about 500mg/kg, about 100 mg/kg to about 400 mg/kg, and about 200 mg/kg to about400 mg/kg), depending on the factors mentioned above. Typically, theclinician will administer an active inhibitor until a dosage is reachedthat results in improvement in or, optimally, elimination of, one ormore symptoms of the treated disease or condition. The progress of thistherapy is easily monitored by conventional assays. One or more agentprovided herein may be administered together or at different times(e.g., one agent is administered prior to the administration of a secondagent). One or more agent may be administered to a subject usingdifferent techniques (e.g., one agent may be administered orally, whilea second agent is administered via intramuscular injection orintranasally). One or more agent may be administered such that the oneor more agent has a pharmacologic effect in a subject at the same time.Alternatively, one or more agent may be administered, such that thepharmacological activity of the first administered agent is expiredprior the administration of one or more secondarily administered agents(e.g., 1, 2, 3, or 4 secondarily administered agents).

Antibodies of the invention (and adjunct therapeutic agent) can beadministered by any suitable means, including parenteral, subcutaneous,intraperitoneal, intrapulmonary, intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, andsubcutaneous administration. In addition, antibodies can be administeredby pulse infusion, particularly with declining doses of the antibody.Dosing can be by any suitable route, e.g., by injections, such asintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic.

The location of the binding target of an antibody used in the inventioncan be taken into consideration in preparation and administration of theantibody. When the binding target is an intracellular molecule, certainembodiments of the invention provide for the antibody or antigen-bindingfragment thereof to be introduced into the cell where the binding targetis located. In one embodiment, an antibody of the invention can beexpressed intracellularly as an intrabody. The term “intrabody,” as usedherein, refers to an antibody or antigen-binding portion thereof that isexpressed intracellularly and that is capable of selectively binding toa target molecule, as described in Marasco, Gene Therapy 4:11-15, 1997;Kontermann, Methods 34:163-170, 2004; U.S. Pat. Nos. 6,004,940 and6,329,173; U.S. Patent Application Publication No. 2003/0104402, and PCTPublication No. WO 03/077945. Intracellular expression of an intrabodyis effected by introducing a nucleic acid encoding the desired antibodyor antigen-binding portion thereof (lacking the wild-type leadersequence and secretory signals normally associated with the geneencoding that antibody or antigen-binding fragment) into a target cell.Any standard method of introducing nucleic acids into a cell may beused, including, but not limited to, microinjection, ballisticinjection, electroporation, calcium phosphate precipitation, liposomes,and transfection with retroviral, adenoviral, adeno-associated viral andvaccinia vectors carrying the nucleic acid of interest.

In another embodiment, internalizing antibodies are provided. Antibodiescan possess certain characteristics that enhance delivery of antibodiesinto cells, or can be modified to possess such characteristics.Techniques for achieving this are known in the art. For example,cationization of an antibody is known to facilitate its uptake intocells (see, e.g., U.S. Pat. No. 6,703,019). Lipofections or liposomescan also be used to deliver the antibody into cells. Where antibodyfragments are used, the smallest inhibitory fragment that specificallybinds to the binding domain of the target protein is generallyadvantageous. For example, based upon the variable-region sequences ofan antibody, peptide molecules can be designed that retain the abilityto bind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology (see, e.g.,Marasco et al., Proc. Natl. Acad. Sci. U.S.A. 90:7889-7893, 1993).

Entry of modulator polypeptides into target cells can be enhanced bymethods known in the art. For example, certain sequences, such as thosederived from HIV Tat or the Antennapedia homeodomain protein are able todirect efficient uptake of heterologous proteins across cell membranes(see, e.g., Chen et al., Proc. Natl. Acad. Sci. U.S.A. 96:4325-4329,1999).

When the binding target is located in the brain, certain embodiments ofthe invention provide for the antibody or antigen-binding fragmentthereof to traverse the blood-brain barrier. Certain neurodegenerativediseases are associated with an increase in permeability of theblood-brain barrier, such that the antibody or antigen-binding fragmentcan be readily introduced to the brain. When the blood-brain barrierremains intact, several art-known approaches exist for transportingmolecules across it, including, but not limited to, physical methods,lipid-based methods, and receptor and channel-based methods.

Physical methods of transporting the antibody or antigen-bindingfragment across the blood-brain barrier include, but are not limited to,circumventing the blood-brain barrier entirely, or by creating openingsin the blood-brain barrier. Circumvention methods include, but are notlimited to, direct injection into the brain (see, e.g., Papanastassiouet al., Gene Therapy 9:398-406, 2002), interstitialinfusion/convection-enhanced delivery (see, e.g., Bobo et al., Proc.Natl. Acad. Sci. U.S.A. 91:2076-2080, 1994), and implanting a deliverydevice in the brain (see, e.g., Gill et al., Nature Med. 9:589-595,2003; and Gliadel Wafers™, Guildford Pharmaceutical). Methods ofcreating openings in the barrier include, but are not limited to,ultrasound (see, e.g., U.S. Patent Publication No. 2002/0038086),osmotic pressure (e.g., by administration of hypertonic mannitol(Neuwelt, E. A., Implication of the Blood-Brain Barrier and itsManipulation, Volumes 1 and 2, Plenum Press, N. Y., 1989)),permeabilization by, e.g., bradykinin or permeabilizer A-7 (see, e.g.,U.S. Pat. Nos. 5,112,596, 5,268,164, 5,506,206, and 5,686,416), andtransfection of neurons that straddle the blood-brain barrier withvectors containing genes encoding the antibody or antigen-bindingfragment (see, e.g., U.S. Patent Publication No. 2003/0083299).

Lipid-based methods of transporting the antibody or antigen-bindingfragment across the blood-brain barrier include, but are not limited to,encapsulating the antibody or antigen-binding fragment in liposomes thatare coupled to antibody binding fragments that bind to receptors on thevascular endothelium of the blood-brain barrier (see, e.g., U.S. PatentApplication Publication No. 2002/0025313), and coating the antibody orantigen-binding fragment in low-density lipoprotein particles (see,e.g., U.S. Patent Application Publication No. 2004/0204354) orapolipoprotein E (see, e.g., U.S. Patent Application Publication No.2004/0131692).

Receptor and channel-based methods of transporting the antibody orantigen-binding fragment across the blood-brain barrier include, but arenot limited to, using glucocorticoid blockers to increase permeabilityof the blood-brain barrier (see, e.g., U.S. Patent ApplicationPublication Nos. 2002/0065259, 2003/0162695, and 2005/0124533);activating potassium channels (see, e.g., U.S. Patent ApplicationPublication No. 2005/0089473), inhibiting ABC drug transporters (see,e.g., U.S. Patent Application Publication No. 2003/0073713); coatingantibodies with a transferrin and modulating activity of the one or moretransferrin receptors (see, e.g., U.S. Patent Application PublicationNo. 2003/0129186), and cationizing the antibodies (see, e.g., U.S. Pat.No. 5,004,697).

Antibody compositions used in the methods of the invention areformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody need not be, but isoptionally formulated with one or more agent currently used to preventor treat the disorder in question. The effective amount of such otheragents depends on the amount of antibodies of the invention present inthe formulation, the type of disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody (when used alone or in combination with other agents) willdepend on the type of disease to be treated, the type of antibody, theseverity and course of the disease, whether the antibody is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1mg/kg-10 mg/kg) of antibody can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the antibody would be in the range fromabout 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof)may be administered to the patient. Such doses may be administeredintermittently, e.g., every week or every three weeks (e.g., such thatthe patient receives from about two to about twenty, or, e.g., about sixdoses of the antibody). An initial higher loading dose, followed by oneor more lower doses may be administered. An exemplary dosing regimencomprises administering an initial loading dose of about 4 mg/kg,followed by a weekly maintenance dose of about 2 mg/kg of the antibody.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

F. Activation of Neuron or Axon Degeneration

The invention also includes methods for activating or increasing neuronor axon degeneration. This may be accomplished by use of an activator oragonist of one or more of the targets listed in Table 2, above (with theexception of adenylyl cyclase, with respect to which an inhibitor can beused to activate neuron or axon degeneration). Thus, agonists of dualleucine zipper-bearing kinase (DLK), glycogen synthase kinase 3β(GSK3β), p38 mitogen-activated protein kinase (p38 MAPK), epidermalgrowth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K),cyclin-dependent kinase 5 (Cdk5), c-Jun N-terminal kinase (JNK),BCL2-associated X protein (Bax), Ih channel, andcalcium/calmodulin-dependent protein kinase kinase (CaMKK) can be usedin methods of activating or increasing axon degeneration. Such agonistsmay be identified or characterized in assays of axon degeneration, asdescribed herein. Thus, for example, a candidate agonist can be presentin medium in which neurons are cultured, in the presence of nerve growthfactor, and assessed for its effect on degeneration. Agonists of neuronor axon degeneration can be used in the prevention and treatment ofdiseases or conditions including epilepsy and autism, as well as anydisease or condition that may be characterized by the failure of anatural process of axon pruning and degeneration. The agonists may beformulated and administered to subjects in need of such treatment usingmethods such as those described in section E, above.

G. Articles of Manufacture

In another aspect of the invention, an article of manufacture (e.g., apharmaceutical composition or kit) containing materials useful for thetreatment or prevention of the disorders and conditions described aboveis provided. The article of manufacture includes a container and a labelor package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, etc. Thecontainers may be formed from a variety of materials such as glass orplastic. The container holds a composition that is by itself or whencombined with another composition effective for treating or preventingthe condition and may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the composition is an inhibitor of the invention. The label orpackage insert indicates that the composition is used for treating thecondition of choice. Moreover, the article of manufacture may include(a) a first container with a composition contained therein, wherein thecomposition includes an inhibitor of the invention; and (b) a secondcontainer with a composition contained therein, wherein the compositionincludes a further therapeutic agent. The article of manufacture in thisembodiment of the invention may further include a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further include a second (or third) container including apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

Further details of the invention are illustrated by the followingnon-limiting examples.

H. Examples

As discussed above, neuron or axon degeneration is a common feature ofmany neurodegenerative diseases and also occurs as a result of injury ortrauma to the nervous system. The mechanisms that regulate this activeprocess, however, are just beginning to be understood. Based on studiesemploying different models of neuron or axon degeneration, it appearsthat different mechanisms may be involved in this process, dependingupon the nature of the disorder or injury. To further characterize theevents that regulate neuron or axon degeneration, a library of smallmolecule compounds targeting known signaling pathways in models ofneuron or axon degeneration were screened. Multiple signaling pathwayswere identified as being necessary for neuron or axon degeneration.Notably, a number of kinases were identified as mediators of neuron oraxon degeneration, and further mechanistic studies localized thefunction of distinct kinases to either the axonal or cell bodycompartments. These pathways were also studied in other degenerationparadigms with similar results, suggesting common molecular mechanismsleading to neuron or axon self-destruction. These experiments aredescribed in the following Examples.

Example 1

Models of Neuron or Axon Degeneration

Three models of neuron or axon degeneration, which were introduced abovein connection with descriptions of assays that can be used to identifyand characterize inhibitors of neuron or axon degeneration, as describedherein, were used in the experiments described below, including anti-NGFantibody, serum deprivation/KCl reduction, and rotenone treatmentassays.

As described above, treatment of cultured nerves with NGF results inproliferation of axons, while treating such nerves with anti-NGFantibodies results in axon degeneration (FIG. 1). Treatment of neuronswith anti-NGF antibodies leads to several different morphologicalchanges that are detectable by microscopy. For example, varicositiesformed in nerves cultured with anti-NGF antibodies before axonfragmentation and, in some cases, appeared to burst (FIG. 2). In anotherexample, axons cultured with anti-NGF antibodies lacked elongatedmitochondria and showed accumulation of mitochondria in varicosities,suggesting that axon transport is blocked. A significant number ofmitochondria were still active within hours of axon fragmentation (FIG.3). A further example relates to cytoskeletal disassembly. Afteraxotomy, the mictrotubule network disassembles prior to the actin andneurofilament networks. In contrast, in the NGF withdrawal model, themicrotubule network was not disassembled before the actin orneurofilament network (FIG. 4).

Another model of axon degeneration is Wallerian degeneration, which isinduced by the occurrence of a lesion in the axon that separates it fromthe cell body (FIG. 5) (see, e.g., Raff et al., Science296(5569):868-871, 2002). In Wallerian Degeneration Slow (Wld^(S))mutants, axon degeneration was significantly delayed, as compared towild type controls (FIG. 5; Araki et al., Science 305(5686):1010-1013,2004). The effect of the Wld^(S) mutant is evidence of the existence ofan axonal self-destruction mechanism, which is weakened in the Wld^(S)mutant. Wld^(S) protected axons after NGF withdrawal, but did notprevent apoptosis.

Additional information concerning the anti-NGF antibody, serumdeprivation/KCL reduction, and rotenone treatment assays is provided inExamples 2-4 (anti-NGF antibody), 7 (serum deprivation/KCl reduction),and 8 and 9 (rotenone treatment).

Example 2

Screen for Inhibitors of Neuron or Axon Degeneration

The anti-NGF antibody model described above in Example 1 was used in ascreen for inhibitors of neuron or axon degeneration. A library of morethan 400 small molecules (Tocris Bioscience) was tested to see which, ifany, modulate degeneration observed in the presence of anti-NGFantibodies.

Methods and Materials

Mouse E13.5 embryos were dissected and placed into L15 medium(Invitrogen). The spinal cord was dissected out from the embryos withDRGs attached. The spinal cords with DRGs attached were placed into L15medium+5% goat serum (Gibco) on ice. The DRGs were removed using atungsten needle and the remaining spinal cord was disposed of. Eightwell slides were filled with N3-F12 solution (23 ml Ham's F12, 1 ml N3supplement, and 1 ml 1 M glucose) to which was added 25 ng/ml NGF(Roche). (N3 supplement was made by dilution of N3 100× concentrate,which was made by mixing the following ingredients, in the followingorder: 5.0 ml Hank's buffered saline solution (HBSS; Ca, Mg free;Invitrogen), 1.0 ml bovine serum albumin (10 mg/ml in HBSS=150 μm), 2.0ml Transferrin (T1147-1G, human, 100 mg/ml in HBSS=1.1 mM), 1.0 mlsodium selenite (S9133-1MG, 0.01 mg/ml in HBSS=58 μM), 0.4 ml putrescinedihydrochloride (P5780-5G, 80 mg/ml in HBSS=500 mM), 0.2 ml progesterone(P8783-5G, 0.125 mg/ml in absolute ethanol=400 μM), 0.02 mlcorticosterone (C2505-500MG, 2 mg/ml in absolute ethanol=5.8 mM), 0.1 mltriiodothyonine, sodium salt (T6397-100MG, 0.2 mg/ml in 0.01 N NaOH=300μM), 0.4 ml insulin (I6634-250MG, bovine pancreas, 241 U/mg, 25 mg/ml in20 mM HCl=4.4 mM), for a total volume of 10.02 ml (can be stored at −20°C.). An N3 supplement stock was made by combining the following: 10 mlPen/Strep (100×, Gibco), 10 ml glutamine (200 mM, Gibco), 10 ml MEMvitamins (100×, Gibco), 10 ml N3 concentrate (100×, see above), for atotal volume of 40 ml. The mixture was filter sterilized using a 0.22 μmfilter, and 1-2 ml aliquots were stored at −20° C.).

The DRGs were sectioned into halves, and placed in the center of eachwell of an 8-chamber slide (BD Biocoat PDL/Laminin coated glass, BectonDickinson). The DRGs were permitted to attach to the slide at roomtemperature for 5-10 minutes, followed by an overnight incubation at 37°C. Inhibitors of choice were added at a concentration of 100 μM (toprow) or 10 μM (bottom row) 1 hour before adding anti-NGF antibodies. Theanti-NGF antibodies were added to the right half of the slide (4 wells)at a concentration of 25 μg/ml. After incubation for 20 hours at 37° C.,the slides were fixed with 30% sucrose/8% paraformaldehyde (PFA) byadding 250 μl of the fix solution directly to the 250 μl of culturemedium. (To make the 30% sucrose/8% PFA solution, the followingingredients were added to a 600 ml beaker including a stir bar: 250 ml16% PFA (cat#15710-S, Electron Microscopy Sciences), 50 ml 10×PBS pH7.4, and 150 g sucrose. The solution was mixed under low heat untildissolved, and then 6-8 drops of 1 M NaOH were added to bring the pH to7.4. The volume was then brought to 500 ml with water in a graduatedcylinder. The solution was mixed well, placed in aliquots, and frozen.)The slides were fixed for 30 minutes, followed by washing once with PBS.All cells were labeled with an actin stain (Alexa-568 conjugatedphalloidin; 1:40; Invitrogen), a membrane dye (DiO; 1:200; Invitrogen),and a DNA stain (Hoechst 33258; 1:10,000; Invitrogen) in 0.1% Triton and1% goat serum for 2 hours at room temperature. The stain solution wasremoved and the slides were washed 1× with PBS, and coverslipped with130 μl of mounting medium (Fluoromount G; Electron Microscopy Sciences)and 24×60 mm no. 1 coverslips (VWR).

Results

In these experiments, control neurons (as visualized by actin staining)underwent significant degeneration upon NGF withdrawal. In contrast, inthe presence of certain small molecules, axon integrity was maintainedupon NGF withdrawal. FIGS. 6-10 show the results of these studies for aproteasome inhibitor and a GSK inhibitor (FIG. 6), a p38 MAPK inhibitorand an adenylyl cyclase activator (FIG. 7), a transcription inhibitorand an EGFR kinase inhibitor (FIG. 8), a JNK inhibitor and a Bax channelblocker (FIG. 9), and an Ih channel blocker and a CaMKK inhibitor (FIG.10). Examples of specific compounds found to protect againstneurodegeneration, and corresponding targets, are shown in Table 5,which is similar to Table 2, above, except that it further includes acolumn with comments concerning observations made with respect to someof the compounds.

TABLE 5 Target Compounds Comments Proteasome MG132 GSK3β SB 415286 GSK3βinhibitor I GSK3β inhibitor VII GSK3β inhibitor VIII GSK3β inhibitor XIILithium Chloride P38 MAPK SB 202190 SB 239063 SB 239069 Filopodiaenriched growth cones present SB 203580 SB 203580 HCI Partial EGFRK AG556 AG 555 AG 494 PD168393 Tyrphostin B44 Partial Tyrphostin B42/AG 490PI3K LY294002 (Calbiochem. Cat. No. 440202) Cdk5 Roscovitine Adenylylcyclase Forskolin NKH 477 Partial Transcription Actinomycin D JNKSP600125 Bax Channel Bax Channel Blocker Cell bodies detach Ih ChannelZD7288 CAMK STO-609 Partial Protein Anisomycin Synthesis Cycloheximide

Example 3

Characterization of Inhibitors with Respect to Timing of Addition afterGrowth Factor Withdrawal

Studies were conducted to assess the activities of the inhibitors whenadded at various time points after NGF withdrawal. In particular,experiments were carried out to determine the latest time-point afterthe start NGF-deprivation at which candidate kinases can be inhibited tostop axon degeneration.

Methods and Materials

Primary cells used in this study were Charles River CD-1 E13 Dorsal RootGanglia (DRG). The cells were maintained in N3/F12 (+25 ng/ml NGF)medium (Ham's F12 (23 ml), N3 supplement (1 ml), glucose (1 ml of 1 Mglucose stock), 25 ng/ml Nerve Growth Factor 2.5 S, mouse (Roche11362348001) in Ham's F12 (stock: 50 μg/ml-80° C.), filter-sterilizedprior to use). The experiments also employed BD Biocoat PDL/Laminincoated glass 8 well chamber slides (BD 354688), and 24×60 mm No. 1coverslips (VWR 48393 106).

Embryos were dissected in L15 medium (the dissection tools were soakedin 70% isopropanol, and dishes were set up on ice: 10 cm dishes (4embryos per dish) and one 6 cm dish for spinal cords). E13.5 spinalcords were extracted into DMEM+10% FBS. >128 DRGs were detached from thespinal cord and sectioned into halves with a tungsten needle. 8 wellslides were filled with 250 μl N3/F12 (25 ng/ml NGF). Sectioned DRGs (˜4per well) were placed in a 5 μl volume. Attachment was allowed for ˜10minutes at room temperature, and then the DRGs were incubated at 37° C.overnight. The following conditions were used to test the inhibitors. 25μg/ml anti-NGF antibody was added at 22 hours. Inhibitors were added atT=0, 1, 3, 6, 9, and 12 hours after anti-NGF addition (1.25 μlinhibitors added; 5 μl aliquot from mass mix into −20° C.; 18 μl in 12μl DMSO; 6 μl in 24 μl DMSO; 18 μl in 12 μl DMSO; 9 μl in 21 μl DMSO; 6μl in 24 μl DMSO). Mixing was done by addition of the inhibitors andlight shaking. The controls were +/−Anti-NGF (+1.25 μl DMSO), and theexperiments were done in duplicate.

After 25 hours, 250 μl 30% sucrose/8% PFA was added to the 250 μl ofculture medium for 30 minutes. Slides were washed with 1×PBS (stop: 4°C.). Immunofluorescence was carried out as follows. First, blocking wascarried out in 5% BSA/0.2% Triton for 30 minutes at room temperature.Primary antibodies (Tuj1 (1:1,000)) were incubated with the slidesovernight in 2% BSA at 4° C. Slides were washed with 1×PBS and add asecondary antibody (Goat anti-mouse 488; 1:200) was added. Slides wereincubated with the secondary antibody for 1 hour at room temperature inthe dark. Slides were washed in 1:10,000 Hoescht in PBS for 10 minutes,washed in PBS in a glass copland for 5 minutes, smacked on a towel tomildly dry, coverslipped with 200 μl of fluoromount G, and stored at 4°C. Pictures were taken all at the same settings (i.e., exposure times).The rate of degeneration was assessed based on detachment of axons fromcell bodies as well.

Results

These studies show that the inhibitors are protective even when addedseveral hours (3, 6, 9, and 12 hours) after NGF withdrawal (FIG. 11).Axon score is a score of axon health on a scale from 1 to 10 (0=lookslike anti-NGF control; 10=looks like NGF control; 5=looks like kinaseinhibitor when added at 0 hours. The inhibitors used in these studiesare listed in Table 6.

TABLE 6 Anti-NGF Inhibitors (stock: 10 mM in DMSO) Cell Body AxonTarget/Action Inhibitor Concentration Compartment Compartment 1) GSK3inhibitor SB415286 30 μM Yes (5)   No (5) 2) EGFR kinase AG555 10 μM No(4) Yes (4) inhibitor 3) p38 MAPK inhibitor SB239063 30 μM No (3) Yes(3) 4) CAMKK inhibitor STO-609 15 μM No (1) Yes (1) 5) JNK inhibitorSP600125 10 μM Yes (1)  Yes (1)

Example 4

Characterization of Compounds with Respect to Localized Degeneration

Campenot chambers were used to further analyze compounds identified asinhibiting axon degeneration in Example 2. Campenot chambers allow theseparation of somal and axonal environments, and permit the induction oflocalized degeneration (see FIG. 12 and, e.g., Zweifel et al., Nat. Rev.Neurosci. 6(8):615-625, 2005). In such chambers, axon degeneration islocalized and proceeds without apoptosis (FIGS. 13 and 14).

Materials and Methods

Teflon dividers (Tyler Research) were cleaned by washing in water andwiping them clean of any residual grease. Dividers were then soaked inNochromix (Godax Laboratories)/sulfuric acid overnight, rinsed fivetimes in distilled and autoclaved water (SQ water), boiled for 30minutes, and then air-dried before use.

Mouse laminin (5 μg/ml in sterile filtered water; Invitrogen) was addedto PDL coated 35 mm dishes (BD Biosciences) and they were incubated for1 hour at 37° C., followed by two rinses in SQ water. The dishes werevacuum-dried and then air-dried in a laminar flow hood for 15 minutes.Prepared dishes were then scored with a pin rake (Tyler Research). Fiftymicroliters of NBM+MC solution containing NGF was applied across theresulting score tracks (The solution was made as follows: 1750 mg ofmethycellulose was combined with 480 ml of Neurobasal (Invitrogen), towhich was added 4.5 ml penicillin/streptomycin, 7.5 ml L-glutamine, 10ml B-27 serum-free supplement (Invitrogen); the solution was mixed forone hour at room temperature, overnight at 4° C., and one further hourat room temperature; the solution was then filter sterilized, and 50ng/ml NGF (Roche) was added prior to use). High vacuum grease (VWR) wasadded to each Teflon divider under a dissection scope. The laminincoated PDL dishes were inverted and dropped onto the Teflon divider,with additional pressure added by use of a toothpick in thenon-track-containing regions. Dishes were incubated for 1 hour at 37° C.Five hundred microliters of NBM+MC (50 ng/ml NGF) solution was added toeach of the side compartments, and a grease barrier was added in frontof the center cell slot.

Free E13.5 spinal cords were dissected from mouse embryos and placedinto NBM+MC (25 ng/ml NGF) solution. DRGs were detached from the spinalcord with a tungsten needle. An NBM+MC-lubricated P200 pipette was usedto move DRGs into a 1.5 ml tube. DRGs were pelleted with a tabletopcentrifuge for 30 seconds. The supernate was discarded and 0.05%Trypsin/EDTA (cold) was added. The pellet was resolubilized with apipette and incubated at 37° C. for 15 minutes with constant agitation(650 RPM). The sample was again centrifuged and the supernate discarded.The pellet was resuspended in warm NBM+MC (50 ng/ml NGF) solution andtriturated with a flamed glass pipette 20 times, followed by triturationwith a fire-bored glass pipette another 20 times. The samples were againcentrifuged and the resulting pellets were resuspended in 0.5 ml NBM+MC(50 ng/ml NGF) solution. The cells were diluted to a final concentrationof 2.5×10⁶ cells/ml. The cell suspensions were loaded into a 1 mlsyringe with a 22 gauge needle. The center slot of the Campenot dividerwas filled using the syringe (to a volume of at least 50 μl). TheCampenot chamber was incubated overnight at 37° C. 2.5 ml NGF+MC (50ng/ml NGF) solution was added to the center compartment and the greasegate was removed. The outer medium (cell body compartment) was replacedafter three days with 2.5 ml NBM+MC medium (with 25 ng/ml NGF). Afterfive days in culture, one distal compartment was washed three times withwarmed NBM+MC (no NGF) solution. After the third wash, 500 μl NBM+MC (noNGF) solution was added to the axon compartment in combination witheither 0.5% DMSO or an inhibitor. The cell body compartment was replacedwith 2.5 ml NBM+MC medium (with 25 ng/ml NGF) containing either 0.5%DMSO or inhibitor.

After 28 hours of anti-NGF antibody treatment, 8% PFA/30% sucrosesolution (see above) was added directly to the culture medium at a 1:1dilution and incubated for 30 minutes. The Teflon divider was removedafter the first 15 minutes of addition. The system was washed once with2.5 ml PBS prior to immunostaining. Neurons were blocked in 5% BSA/0.2%triton in PBS for 30 minutes. The primary antibody Tuj1 (Covance) wasadded to a final dilution of 1:1000 in PBS containing 2% BSA andincubated overnight at 4° C. The dish was washed once with PBS. Thesecondary antibody (Alexa 488 goat anti-mouse antibody (Invitrogen)) wasadded at a final dilution of 1:200 in 2% BSA in PBS and incubated forone hour at room temperature. The dish was washed twice with PBS, and a22×22 mm coverslip (VWR) was added with 350 μl of fluoromount G(Electron Microscopy Sciences). The neurons were visualized by use of afluorescence microscope.

Results

As described above, E13.5 DRGs were isolated and grown for 5 days in aCampenot chamber. Fifty μg/ml anti-NGF was added to the experimentalaxon compartment, with an inhibitor added either to the axon compartment(together with the NGF antibody), or the cell body compartment, and theaxons were allowed to degenerate for 28 hours. Another axon compartmentwas maintained in NGF as a control. As shown in FIGS. 15 and 16, whencell bodies were exposed to SB415 (GSKi) or Act D (transcriptioninhibitor), or when axons were exposed to AG555 (EGFRi) or SB239 (p38i),axons deprived of NGF did not degenerate. In contrast, SB415 (GSKi) orAct D (transcription inhibitor) in the NGF-deprived axon compartment, orAG555 (EGFRi) or SB239 (p38i) treatment in the cell body compartment,failed to prevent degeneration, suggesting that signaling in local axondegeneration is not limited to the axon segment being lost; someinhibitors are most effective when applied to the cell body, and othersto the axon. Quantification of these results is shown in FIG. 17. Table7 provides a summary of axon degeneration data from Campenot chambers.

TABLE 7 Inhibitors in Axon vs. Cell Body Compartment Cell Body Concen-Compart- Axon Target/Action Inhibitor tration ment Compartment CellularIC₅₀ Transcription Actinomycin D 15 μM Yes (4)  No (4) inhibitor Proteinsynthesis Cycloheximide  5 μM Yes (2)  No (2) inhibitor Proteasomeinhibitor MG132 0.5 μM  Yes (1)  No (1) GSK3 inhibitor SB415286 30 μMYes (5)  No (5)  10 μM^(a) GSK3 inhibitor 30 μM Yes (2)  No (2) XIAR-A014418 20 μM Yes (1)  No (1) Ih channel blocker ZD7288  5 μM Yes (3) No (3) ErbB kinase AG555 10 μM  No (4) Yes (4) inhibitor PD168393  5 μM No (1) Yes (1)   5 nM^(b)  100 nM^(c) p38 MAPK inhibitor SB239063 30 μM No (3) Yes (3) 0.35 μM^(d)  5 μM  No (1) Yes (1) CAMKK inhibitorSTO-609 15 μM  No (1) Yes (1)   3 μM^(e) Adenylyl cyclase Forskolin  3μM Yes (2) Yes (2) activator JNK inhibitor SP600125  5 μM Yes (1) Yes(1) (Note: IC₅₀ information in Table 7 is from published sources andsome values were generated with non-neuronal cell types) ^(a)Cross etal., J. Neurochem. 77(1): 94-102, 2001; total GSK-3 activity by in vitropeptide assay in cerebellar granule neurons. ^(b)Fry et al., Proc. Natl.Acad. Sci. U.S.A. 95(20): 12022-12027, 1998; heregulin induced tyrosinephosphorylation in MDA-MB-453 cells. ^(c)Bose et al., Proc. Natl. Acad.Sci. U.S.A. 103(26): 9773-9778, 2006; protein tyrosine phosphorylationin 3T3-Her2 cells. ^(d)Barone et al., J. Pharmacol. Exp. Ther. 296(2):312-321, 2001; LPS-induced TNF-alpha production in human monocytes.^(e)Tokumitsu et al., J. Biol. Chem. 277(18): 15813-15818, 2002; CaMKKactivity after ionomycin stimulation in transfected HeLa cells.

A model based on data from the screens described above is shown in FIG.18. Preliminary studies showed that cell bodies appeared smaller whenNGF was removed from the axon compartment (FIG. 19), and that many ofthe neurons locally deprived of NGF were cleaved caspase-3-positive andshowed nuclear condensation (FIG. 20). Further studies show that GSK3(FIG. 21) and JNK (FIG. 22) activities peak 6 hours after NGFwithdrawal.

Example 5

Characterization of Compounds with Respect to Particular Phenotypes ofNeuron Degeneration

With compounds identified above as being cell body inhibitors (SB415(GSKi) and Act D (transcription inhibitor)) applied to the cell body,and compounds identified as axon inhibitors (AG555 (EGFRi) and SB239(p38i)) applied to axons, locally deprived axons were observed withrespect to formation of varicosities using the Campenot chamber assaydescribed above. A large number of axons with varicosities, as well asfragmented axons, were observed with the cell body inhibitors (FIG. 23).The axon inhibitors showed fewer varicosities, and the axons appeared toproceed straight to fragmentation upon treatment with those inhibitors(FIG. 23). These observations indicate that EGFR and p38 may be upstreamof the formation of varicosities. Mitochondrial dysfunction and blockadeof axon transport were studied with GSK, EGFR, and p38 inhibitors. Asshown in FIG. 24, functional mitochondria were observed, but none wereelongated.

Inhibiting GSK in an axon degeneration paradigm where signaling throughthe cell body does not occur was also studied. Fifty μM SB415 slightlydelayed degeneration after lesion, so inhibiting GSK3 has a directeffect on the axon, most likely through stabilization of the axonalmicrotubule network. This effect likely contributes to the delay in axondegeneration observed after global NGF withdrawal, but the role of GSK3bin the cell body appears to be more significant (FIG. 25). Additionally,protection of axons after global NGF withdrawal appears to beindependent of the role of GSK in neuronal death since the GSK inhibitorblocked axon degeneration but did not block cell death (FIG. 26).

Example 6

Protection Against Peripheral Neuropathy in Patients

Tarceva® (erlotinib) is an EGFR kinase inhibitor. Treatment of patientswith paclitaxel and other chemotherapeutic agents is known to lead toperipheral neuropathy (reviewed in Wilkes, Semin. Oncol. Nurs.23(3):162-173, 2007). Patients treated with a combination of Tarceva®and paclitaxel showed significantly less peripheral neuropathy (p=0.012;Fisher's exact), as compared to a placebo+paclitaxel group (16.3% vs.26.4% of patients; first 400 patients enrolled; analysis of commonadverse events).

Example 7

Analysis of EGFR Kinase Levels in an Amyotrophic Lateral Sclerosis (ALS)Model

Amyotrophic lateral sclerosis (“ALS”) is a severely debilitating andfatal neurodegenerative disease, the pathology of which is characterizedby loss of motor neurons. Similar pathology occurs in mouse models ofALS containing mutations in the Superoxide Dismutase (“SOD”) gene(SOD(G93A)). Phosphorylation of the EGF receptor is associated withaxonal degeneration. EGFR levels are altered in SOD(G93A) mice, and anexperiment was performed to determine whether phosphorylated EGFR levelsare also altered in SOD(G93A) mice.

Spinal cords from terminal SOD(G93A) and non-transgenic littermates werecryosectioned (20 μm thick) onto slides and stored at −80° C. Slideswere thawed to room temperature and hydrated in PBS twice for 5 minutesper rinse. Slides were blocked in hydrogen peroxide (0.3% in PBS) for 5minutes, and then were rinsed twice in PBS for 5 minutes per rinse.Slides were washed twice in PBS-T (PBS containing 0.1% Triton X100) for10 minutes per wash. Slides were blocked in PBS containing 5% BSA and0.3% Triton X100 for about 1 hour at room temperature. A rabbitmonoclonal anti-phosphorylated EGFR primary antibody (Novus Biologicals)was diluted 1:500 in 1% BSA and 0.3% Triton X100 in PBS and incubatedwith the slides overnight at 4° C. The slides were washed four times inPBS-T for 10 minutes per wash. A biotinylated goat anti-rabbit secondaryantibody (Vector Labs) was diluted 1:300 in 1% BSA and 0.3% Triton X100in PBS and incubated with the slides for 30 minutes-1 hour. The slideswere washed four times in PBS-T for 5 minutes per wash. Washed slideswere incubated in Avidin-biotin complex solution (Vector Laboratories)for 30 minutes at room temperature. The slides were washed four times inPBS-T for 5 minutes per wash. The slides were then incubated with aperoxidase substrate (Diaminobenzidine (DAB); Sigma) until the desiredintensity developed. The slides were rinsed in water, coverslipped, andviewed.

As shown in FIGS. 27 and 28, which show sections of spinal cord stainedwith EGFR antibodies, phosphorylated EGFR levels were increased in theSOD mice, as compared to the control mice. This observation shows thatEGFR kinase inhibition may be beneficial in the treatment of ALS invivo.

Immunohistochemical studies of SOD1 samples were also carried out.Slides were warmed to room temperature, outlined with an ImmEdge pen,hydrated by 2×10 minute washes in PBS at room temperature, and washedfor 2×10 minutes in PBSTx at room temperature. Antibody blocking wascarried out for 1-2 hours at room temperature (5% BSA, 0.3% Tx in PBS;(25 mg BSA in 500 ml PBS+1.5 ml 10% Tx)). Samples were incubated withprimary antibodies overnight at 4° C. in 1% BSA, 0.3% Tx in PBS (NF(mouse, Millipore) 1:200; SMI32 (mouse, Covance) 1:300)). Samples werewashed after primary antibody incubation 4×10 minutes in PBSTx (0.1%) atroom temperature. Secondary antibodies were applied (Molecular Probes,Alexa conjugated; Donkey anti-Mouse Alexa-488; diluted 1:500 in 1% BSA,0.3% Tx in PBS) and samples were incubated for 2 hours at roomtemperature. Samples were washed after secondary antibody incubation2×10 minutes in PBSTx, and 2×10 minutes PBS. Optionally, nuclearcounterstaining was carried out during the second wash by application ofDAPI at 1:10,000 in PBS. Mounting was carried out with Vectashieldmounting medium (Vector Labs).

FIG. 29 shows pEGFR remaining in axons in the SOD ALS model. As shown inthe figure, axon number decreases in SOD1-tg spinal cord, and pEGFRpartially co-localizes with axons in SOD1-tg animals. Thus, axons arelost in SOD1 transgenic mice as compared to non-transgenic controls, andmany of the remaining exons are immunoreactive for pEGFR.

Example 8

Protection of Cerebellar Granule Neurons from Serum Deprivation/KClReduction-Induced Degeneration

Kinase inhibitors (inhibitors of GSK3, JNK, EGFR, p38, and CaMKK) weretested for their capacity to protect cultured cerebellar granule neurons(CGNs) following serum deprivation/KCl reduction. Briefly, CGNs isolatedfrom P7 mouse brain were cultured on PDL- and laminin-coated 96-welltissue culture dishes in medium containing serum and potassium (BasalMedium Eagles including 29 mM KCl and 10% FBS) at 37° C. After 24 hoursin culture, cells were switched to “deprivation” medium (Basal MediumEagles including 5 mM KCl), alone or in combination with various smallmolecule inhibitors as shown in Table 8. After a further 24 hours inculture, the neurons were fixed with 4% paraformaldehyde and stainedwith a neuronal marker (anti-class III β-Tubulin, Covance). Imageacquisition was performed using the ImageXpress automated imaging system(Molecular Devices). The plate set-up used is summarized in Table 8.

TABLE 8 1 2 3 4 5 6 7 8 9 10 11 12 A Positive Control 5 mM KCl + 1 μMPositive Control 5 mM KCl + 1 μM (29KCl + serum) EGFR kinase inhibitor(29 mM KCl + serum) EGFR kinase inhibitor B Negative Control 5 mM KCl +10 μM Negative Control 5 mM KCl + 10 μM (5 mM KCl) EGFR kinase inhibitor(5 mM KCl) EGFR kinase inhibitor C 5 mM KCl + 5 μM 5 mM KCl + 30 μM 5 mMKCl + 5 μM 5 mM KCl + 30 μM JNK inhibitor EGFR kinase inhibitor JNKinhibitor EGFR inhibitor D 5 mM KCl + 10 μM 5 mM KCl + 5 μM 5 mM KCl +10 μM 5 mM KCl + 5 μM JNK inhibitor p38 inhibitor JNK inhibitor p38inhibitor E 5 mM KCl + 20 μM 5 mM KCl + 10 μM 5 mM KCl + 20 μM 5 mMKCl + 10 μM JNK inhibitor p38 inhibitor JNK inhibitor p38 inhibitor F 5mM KCl + 5 μM 5 mM KCl + 30 μM 5 mM KCl + 5 μM 5 mM KCl + 30 μM CAMKKinhibitor p38 inhibitor CAMKK inhibitor p38 inhibitor G 5 mM KCl + 15 μM5 mM KCl + 10 μM 5 mM KCl + 15 μM 5 mM KCl + 10 μM CAMKK inhibitor GSK3inhibitor CAMKK inhibitor GSK3 inhibitor H 5 mM KCl + 30 μM 5 mM KCl +30 μM 5 mM KCl + 30 μM 5 mM KCl + 30 μM CAMKK inhibitor GSK3 inhibitorCAMKK inhibitor GSK3 inhibitor JNK inhibitor = SP600125 CAMKK inhibitor= STO-609 EGFR inhibitor = AG 555 P38 inhibitor = SB 239063 GSK3inhibitor = SB 415286 Columns 1-6: cell density = 5 × 10⁴/well Columns7-12: cell density = 2.5 × 10⁴/well

As shown in FIG. 30, the kinase inhibitors were found to be effective atprotecting the CGNs from degeneration, similar to the findings inExamples 2 and 3 for DRGs.

Example 9

Protection of Hippocampal Neurons from Rotenone-Induced Degeneration

Kinase inhibitors (inhibitors of GSK3, JNK, EGFR, p38, and CaMKK) weretested for their capacity to protect hippocampal neurons fromrotenone-dependent degeneration. The assay was performed as follows.First, hippocampal neurons were dissected. Briefly, E18-E19 embryos wereremoved from anesthetized pregnant rats, placed in Petri dishescontaining cold HBSS (see above), and washed in fresh, cold HBSS.Embryos were removed from their sacs and placed in fresh, cold HBSS, andthe brains were isolated and placed in fresh, cold HBSS using standardtechniques. The hippocampus and cortex were isolated from each embryo,and the brains were cut sagitally in half to separate the hemispheres.Remaining cerebellum/brain stem, olfactory bulbs, non-cortex ventralunderlying tissue, and the meninges were removed. The hippocampi wereremoved from the remaining tissue and transferred to a 1.5 ml tube onice. The cortical tissues were removed to a separate 1.5 ml tube on ice.As much HBSS as possible was removed, and 1% Trypsin (Hyclone)/0.1%DNase (Sigma) in HBSS was added to the remaining tissue in each tube.The tissues were each broken up using a fire-polished pipette. Thetissues were incubated for ten minutes at 37° C., with tapping of thetubes every two minutes. As much solution as possible was removed fromthe tissues, and the tissues were each washed with 0.05% DNase (Sigma)solution. The samples were triturated in 0.05% DNase (Sigma) about 20×with each of the following (1) fire polished pipette at ⅔ bore, and (2)fire polished pipette at ⅓ bore. The samples were permitted to settlefor five minutes, after which the supernatants were collected (thedebris settled and the supernatant was removed with a pipette) and thecells were counted on a hemocytometer.

Nucleofection was performed on the isolated hippocampal neurons.Briefly, the desired number of cells (0.5-1×10⁶ cells per nucleofection)were isolated by centrifugation. As much of the supernatant solution aspossible was removed and the pelleted cells from each tube wereresuspended in 20 μl room temperature nucleofection solution (Amaxa).Each resuspension was added to pre-aliquoted β-actin-GFP “pCAGGS-AFP”DNA (400 ng per run) and tapped gently. The mixtures were transferred tonucleocuvette wells of a 96 well shuttle plate, and the plate wasinserted into the nucleofector device. Neurons were nucleofected using apreset Amaxa program, CU110. Immediately following nucleofection, 100 μlpre-warmed CNBM (10 ml B27 (serum-free supplement; Invitrogen), 5 mls100× Pen-Strep, 5 mls 100× Glutamax, 480 mls NBM (neurobasal medium;Invitrogen)) was added, and the cells were subsequently plated at adensity of 70,000 cells/well in 8-well PDL-coated chamber slides.Neurons were grown for 5-14 days before rotenone addition, with changesof medium every 3-5 days.

Ten μM rotenone (resuspended in DMSO; VWR) was added to neurons in thepresence or absence of vehicle or experimental compounds. 17-18 hoursafter rotenone addition, neurons were fixed in a solution of 4%paraformaldehyde/15% sucrose for 40 minutes at room temperature. Neuronswere washed once with PBS and mouse anti-Tuj1 antibody (Covance) wasadded at a dilution of 1:1000 (in PBS, 0.1% Triton X-100, 2% goat serum)for an overnight incubation at 4° C. Neurons were washed 4× with PBS andgoat anti-mouse antibody conjugated to Alexa-568 (Alexa) was added at adilution of 1:200 (in PBS, 2% goat serum) for 30 minutes. Neurons wereagain washed 4× with PBS and mounted on slides in Vectashield mountingmedium (Vector Labs). All neurons were visualized with Tuj1, andindividual neurons were visualized with GFP.

As shown in FIG. 31, the kinase inhibitors were found to protecthippocampal neurons from rotenone-dependent degeneration. Similarstudies were carried out with cortical neurons, and showed that corticalneurons are also protected from rotenone-dependent degeneration bykinase inhibitors (FIG. 32).

Example 10

Visualization of ErbB Receptor on Axons

To visualize ErbB receptor expression on axons, DRGs were cultured asdescribed above for the screen. After 24 hours of growth, cells werefixed by adding 8% PFA/30% sucrose to culture medium at a 1:1 ratio for30 minutes and washed 1× with PBS. Fixed cells were incubated in 5% BSAand 0.2% TritonX100 in PBS for 30 minutes, and then incubated overnightin 2% BSA in PBS at 4° C. with the following antibodies: (1) 50 μg/mlanti-EGFR (D1-5, Genentech), (2) 1:500 anti-ErbB2 (Abcam), (3) 24 μg/mlanti-ErbB3 (57.88, Genentech), and (4) 1:500 ErbB4 (Abcam). Cells werewashed 1× with PBS, followed by incubation with a fluorescentlyconjugated secondary antibody (1:200, Invitrogen) at room temperaturefor 30 minutes, washed 1× with PBS containing Hoechst 33258 (1 μg/ml,Invitrogen), followed by a final PBS wash, and coverslipped with 250 μlof Fluoromount G (Electron Microscopy Sciences). As shown in FIG. 33,ErbBs are detected on axons by immunocytochemistry.

Example 11

Characterization of EGFR Expressed on Axons by Use of EGFR Ligands

Experiments were carried out to assess the effects of activation of EGFRby ligands, including EGF.

Materials and Methods

Dorsal Root Ganglia (DRG) immunoblots were prepared as follows. Primarycells used in the experiments were Charles River CD-1 E13 Dorsal RootGanglia (DRG). The cells were maintained in N3/F12 (+25 ng/ml NGF)medium (Ham's F12 (23 ml), N3 supplement (1 ml), glucose (1 ml of 1 Mglucose stock), 25 ng/ml Nerve Growth Factor 2.5 S, mouse (Roche11362348001) in Ham's F12 (stock: 50 μg/ml-80° C.), which wasfilter-sterilized prior to use). The experiments also employed Tritonlysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Triton-X100, 100×Phosphatase inhibitor cocktail I (added fresh), 100× Phosphataseinhibitor cocktail II (added fresh), 1 tablet/10 ml Protease inhibitortablet (added fresh)).

DRG explants were prepared on Day 0. Embryo dissections were carried outin L15 medium (dissection tools were soaked in 70% isopropanol, anddishes were set up on ice: 10 cm dishes (4 embryos per dish) and one 6cm dish for spinal cords). E13.5 spinal cords were extracted intoDMEM+10% FBS. 5×20 DRGs were detached from the spinal cord. 8 wellslides were filled with 250 μl N3/F12 (25 ng/ml NGF)+7 μM cytosinearabinoside. Approximately 20 DRGs were placed in each well (fivetotal), and attachment was allowed for about 10 minutes at roomtemperature. On day 2, media was changed to N3/F12+25 ng/ml NGF. On day3, ErbB ligands were added (1000×; 100 μg/ml; dilute in SQ water 1:10)for 30 minutes to 1 hour (SQ water (control); EGF (ErbB1); Neuregulin-1(ErbB3); Beta-cellulin (ErbB1/4); Epiregulin (ErbB1/4); Anti-NGF (25μg/ml)) and slides were shaken slightly.

Immunobloting was carried out by aspiration of media with a pipette tip,washing twice with ice-cold sterile PBS, and removal of all PBS using anaspirator with a pipette tip or kimwipes. Cells were lysed with 40 μlTriton lysis buffer on ice. Lysate was collected, rotated for 30 minutesat 4° C., spun for 5 minutes at top speed, and supernatant was kept(stop: −80° C.). About 20 μl of sample was loaded. The sample wasprepared by adding 1×SDS sample buffer and NuPAGE reducing agent (10×),and the lysate was boiled. Samples were fractionated by 10% SDS-PAGE (at120 Volts)(run for two membranes) with standards (1 μl MWM; 1 μl and 2μl spinal cord lysate). The gels were blotted using the iBlot program 3(Nitrocellulose), and the blot was cut at 98 kDa. The top membrane wasblocked in 5% BSA in TBST for 1 hour. Primary antibody was placed into 2ml 5% BSA in TBST for 1 hour (Calbiochem anti-P-tyrosine HRP conjugate(PY20) 1:10,000). The blot was washed with TBST for 3×5 minutes anddeveloped with ECL. The bottom membrane was blocked in 5% milk for onehour in PBS. Primary antibodies were placed in 2 ml 5% BSA in TBST 4° C.for two days in vacuum bag. The antibodies used were CS anti-P-Erkrabbit (42/44 kDa) 1:1000; C anti-TUJ1 rabbit (55 kDa) 1:5000 (on aseparate membrane), and SC anti-Erk1 rabbit (42/44 kDa) 1:1000 (on aseparate membrane). The blot was washed in TBST for 3×5 minutes.Secondary antibody was added into 10 ml 5% milk in TBST 1:5000 andincubated for 1 hour at room temperature (Red (680) anti-rabbit). Theblots were washed with TBST for 3×5 minutes, and scanned in PBS withOdyssey. Quantification was carried out by choosing a background methodand standard bands that minimize negative/counter-intuitive values:i.e., top/bottom, right/left, user-defined (user-defined). The lower theintensity of the concentration standard, the fewer negative values.

Results

The goal of these experiments was to see if activation of EGFR by EGFwould be sufficient to drive degeneration. As shown in FIG. 34, EGFR isexpressed on axons. FIG. 35 shows that EGF added to neurons maintainedin NGF does not cause degeneration. To ensure that EGF is capable ofactivating EGFR/ErbBs in the neuronal cultures used in these assays, ERKactivation was assessed (ERK is a downstream target of EGFR). As shownin the graph in FIG. 35, adding EGF increased ERK activity, asdetermined by Western blot. These data show that activating EGFR is notsufficient to drive degeneration.

Example 12

Characterization of EGFR Expressed on Axons by Use of EGFR Ectodomains

Experiments were carried out to assess the effects of EGFR ectodomainson activation of EGFR.

Materials and Methods

Materials used in these studies include BD Biocoat PDL/Laminin coatedglass 8 well chamber slides (BD 354688); L15 medium (Invitrogen11415114); albumin from bovine serum (BSA) (Sigma A7906-500 g); fetalbovine serum (Sigma F2442-100 ml); N3 supplement (see above);Fluoromount G (Electron Microscopy Sciences 17984-25); 24×60 mm No. 1coverslips (VWR 48393 106); monoclonal anti-neuronal class IIIbeta-tubulin (Covance MMS-435P); Nerve Growth Factor 2.5 S, mouse (Roche11362348001) in Ham's F12; and Triton X-100 (Sigma T8787-100 ml).Solutions used in these experiments include 25 ml N3/F12 medium (23 mlof Ham's F12; 1 ml of N3 supplement; 1 ml of 1M glucose; 25 ng/ml ofNGF) and 30% Sucrose/8% PFA (see above).

Ectodomains (R&D Systems) were resuspended in sterile filtered 0.1% BSAin PBS to 1 mg/ml. Mouse E13.5 embryos were dissected out and place intoL15 medium. Using forceps, the ventral region of the embryo was opened,organs removed, ribs cut away, and spinal cord dissected out with DRGsattached. The spinal cords with DRGs attached were placed into L15medium+5% goat serum on ice. DRGs were removed with a tungsten needleand the remaining spinal cord was disposed of. 8 well slides were filledwith N3-F12 plus 25 ng/ml NGF. DRGs were sectioned into halves.Sectioned DRGs were placed in the centers of each well of an 8-chamberslide and DRGs were allowed to attach at room temperature for 5-10minutes. Ectodomain was added to a final concentration of 50 μg/ml inthe top row. Slides were placed in a 37° C. incubator overnight.Ectodomain was added to the bottom row to a final concentration of 50μg/ml. Anti-NGF antibody was added to the wells at a concentration of 25μg/ml. EGFR inhibitor AG555 was added at a concentration of 10 μM as apositive control.

After 24 hours of anti-NGF antibody treatment, 8% PFA/30% sucrose wasadded directly to the culture medium at a 1:1 dilution for 30 minutes.The Teflon divider was removed after the first 15 minutes, and slideswere washed once with PBS.

Slides were blocked in 5% BSA/0.2% Triton for 30 minutes, and incubatedwith primary antibodies (Tuj1 (1:1,000)) overnight in 2% BSA. Slideswere washed with PBS once and secondary antibody (Goat anti-mouse 488;1:200) was added. Slides were incubated for 1 hour at room temperature,washed 2× with PBS, coverslipped with 250 μl of fluoromount G, andstored at 4° C.

Results

In addition to EGF, there are many ligands that can activate EGFR. Totest whether EGFR activation during degeneration is ligand dependent,EGFR ectodomain from R&D Systems was added at a concentration of 50μg/ml, which should be sufficient to bind any free ligands that mayactivate EGFR. The EGFR ectodomain, added either 24 hours prior to, orat the same time as adding anti-NGF, does not block degeneration. Thissuggests EGFR activation during degeneration is likelyligand-independent.

Example 13

The dorsal spinal cord (DSC) explant is another model ofneurodegeneration. DSCs grow out axons over a period of days, but ifthey are not rescued with the addition of a survival factor, they willeventually degenerate. As shown in FIG. 37, Tarceva® (Erlotinib) candelay this degeneration program.

Example 14

A. Inhibition of Dual Leucine Zipper-bearing Kinase

Materials and Methods

DLK Transfection in 293 Cell Line

293 cells were plated at 30% confluence into 3 wells of a 6-well plate.Cells were transfected with either control plasmid DNA, a DNA constructexpressing wild type DLK, or two plasmids, with one expressing wild typeDLK and the other expressing a DLK in which threonine 277 has beenmutated to alanine (T278A), creating a form of the protein withoutactivity (Fugene 6, Roche). Twenty-four hours after transfection, cellswere washed with PBS, scraped off each plate, and transferred to aneppendorf tube. Cells were then spun down and excess media was removed.Pelleted cells were lysed in 20 mM Tris, 150 mM NaCl, 0.1% Triton X-100,and placed at 4° C. for 30 minutes. Samples were then spun to removeinsoluble particles and tested for protein concentration in abicinchoninic acid (BCA) assay (Promega).

Samples were run on a 10% polyacrylamide gel (Invitrogen) andtransferred using an iBlot device (Invitrogen) according to themanufacturer's specifications. Blots were blocked for 1 hour in TBST(Tris-buffered saline+1% Tween 20) with 5% bovine serum albumin (BSA).After blocking, blots were incubated overnight in primary antibodiesdirected against JNK (Cell Signaling Technologies) or the phosphorylatedform of JNK (Cell Signaling Technologies) in TBST+1% BSA. Afterincubation, blots were washed 3 times in TBST and then incubated withHRP-conjugated anti-rabbit secondary antibodies for 1 hour at roomtemperature. Blots were once again washed three times and then incubatedwith ECL (Promega) for 1 minute. Blots were then exposed on film andanalyzed.

Degeneration in DRGs after DLK siRNA

Mouse E13.5 embryos were dissected and placed into L15 medium(Invitrogen). The spinal cord was dissected out from the embryos withDRGs attached. The spinal cords with DRGs attached were placed into L15medium+5% goat serum (Gibco) on ice. The DRGs were removed using atungsten needle and the remaining spinal cord was disposed. Eight-wellslides were filled with N3-F12 solution (23 ml of Ham's F12, 1 ml of N3supplement, and 1 ml of 1 M glucose) to which was added 25 ng/ml NGF(Roche). (N3 supplement was made by dilution of N3 100× concentrate,which was made by mixing the following ingredients, in the followingorder: 5.0 ml Hank's buffered saline solution (HBSS; Ca, Mg free;Invitrogen), 1.0 ml bovine serum albumin (10 mg/ml in HBSS=150 μm), 2.0ml Transferrin (T1147-1G, human, 100 mg/ml in HBSS=1.1 mM), 1.0 mlsodium selenite (S9133-1MG, 0.01 mg/ml in HBSS=58 μM), 0.4 ml putrescinedihydrochloride (P5780-5G, 80 mg/ml in HBSS=500 mM), 0.2 ml progesterone(P8783-5G, 0.125 mg/ml in absolute ethanol=400 μM), 0.02 mlcorticosterone (C2505-500MG, 2 mg/ml in absolute ethanol=5.8 mM), 0.1 mltriiodothyonine, sodium salt (T6397-100MG, 0.2 mg/ml in 0.01 N NaOH=300μM), 0.4 ml insulin (16634-250MG, bovine pancreas, 241 U/mg, 25 mg/ml in20 mM HCl=4.4 mM), for a total volume of 10.02 ml (can be stored at −20°C.)). An N3 supplement stock was made by combining the following: 10 mlPen/Strep (100×, Gibco), 10 ml glutamine (200 mM, Gibco), 10 ml MEMvitamins (100×, Gibco), 10 ml N3 concentrate (100×, see above), for atotal volume of 40 ml. The mixture was filter sterilized using a 0.22 μmfilter, and 1-2 ml aliquots were stored at −20° C.

Neurons were trypsinized using 0.05% trypsin-EDTA (Gibco) at 37° C. for30 minutes, spun down and counted. Two hundred thousand cells wereelectroporated using an Amaxa 96-well nucleofector with 400 ng controlof DLK siRNA (program DC 100), then split equally onto two wells of an8-chamber slide (BD Biocoat PDL/Laminin coated glass, Becton Dickinson).Neurons were permitted to attach to the slide at room temperature for5-10 minutes, followed by incubation for 3 days at 37° C. The anti-NGFantibodies were then added to the right half of the slide (4 wells) at aconcentration of 25 μg/ml. After incubation for 20 hours at 37° C., theslides were fixed with 30% sucrose/8% paraformaldehyde (PFA) by adding250 μl of the fix solution directly to the 250 μl of culture medium. (Tomake the 30% sucrose/8% PFA solution, the following ingredients wereadded to a 600 ml beaker including a stir bar: 250 ml 16% PFA(cat#15710-S, Electron Microscopy Sciences), 50 ml 10×PBS pH 7.4, and150 g sucrose. The solution was mixed under low heat until dissolved.Then 6-8 drops of 1 M NaOH were added to bring the pH to 7.4. The volumewas then brought to 500 ml with water in graduated cylinder. Thesolution was mixed well, placed in aliquots, and frozen).

The slides were fixed for 30 minutes, followed by washing three timeswith PBS. All cells were labeled with a neuron specific β-tubulinantibody (Tuj1 (1:1000) in 2% BSA, 0.1% Triton) at 4° C. overnight. Theprimary antibody was removed and the slides were washed three times withPBS. Slides were incubated with goat Alexa 488 anti-rabbit secondaryantibody (1:500) for one hour followed by three washes in PBS, and thencoverslipped with 130 μl of mounting medium (Fluoromount G; ElectronMicroscopy Sciences) and 24×60 mm No. 1 coverslips (VWR).

Quantitative PCR (qPCR) for DLK

E13.5 DRGs were dissected, electroporated with control siRNAs or siRNAsdirected against DLK, and cultured as described above for a period offive days. RNA from neurons was then isolated using Purify Total RNA byQiagen RNeasy Mini kit, according to the manufacturer's protocols. 10 ngof total RNA from cells treated with control and DLK siRNAs were run intriplicate in quantitative PCR experiments (Qiagen Quantifect SYBR GreenRT-PCR) using DNA primers specific for DLK and GAPDH (control)(CATCATCTGGGTGTGGGAAG (forward primer) and AGTTGCAGCATGAGGGCATTC(reverse primer); SEQ ID NOs: 16 and 17). Amplification curves wereanalyzed and relative RNA concentrations of each sample were calculatedrelative to controls.

Results

The results of these experiments are shown in FIG. 38. Transienttransfection of 293 cells with a plasmid encoding wild type DLK resultedin the phosphorylation and activation of JNK, showing that DLK is akinase upstream of JNK in a signaling cascade. Co-transfection of aplasmid encoding a kinase-dead DLK reduced the levels of JNKphosphorylation. Knockdown of DLK expression via siRNA resulted inprotection of neurons against degeneration as did inhibition of thedownstream kinase JNK. In experiments using cultured neurons transfectedwith siRNAs directed against DLK (Dharmicon), neurons transfected withcontrol siRNAs (as visualized by actin staining) underwent significantdegeneration upon NGF withdrawal as visualized by TuJI staining (FIGS.38 and 39). In contrast, in neurons transfected with siRNAs targetedagainst DLK, axon integrity was maintained upon NGF withdrawal.Knockdown of DLK expression in these experiments was confirmed usingquantitative PCR with primers specific to DLK.

B. DLK Knockdown Protects Against Toxin-Induced Neuronal Cell Death

Experiments were performed to assess whether the above-observedprotective effect of DLK inhibition is more generally protective againstaxon degeneration/neuronal apoptosis caused by other insults, such astoxin exposure. Isolated neurons were exposed to vincristine, a mitoticinhibitor, in the presence or absence of siRNA specific for DLK.

Cortical neurons were isolated using the following procedure. Rat E18cortices devoid of meninges were dissected into ice-cold neurobasalmedia (Invitrogen). Cortices were dissociated in a final concentrationof 0.1% trypsin/PBS (Worthington) for 30 minutes at 37° C. A finalconcentration of 0.1% DNase (Roche) was added to the tube and tissue wasincubated at room temperature for 1 minute. Tissue was washed once withwarm neurobasal media and the tissue was allowed to settle in the tubebefore 1 mL of neurobasal media containing 2% B27 supplement(Invitrogen) was added. Tissue was dissociated by trituration with aP1000 pipet (Rainin). The cells were counted and, for some experiments,transfected with siRNAs using a Biosystem 96-well nucleofector (Amaxa).

Cells were plated in 6- or 8-well chambers or 96-well poly-D-lysinecoated dishes (BD Biosciences) at plated cell densitites of 2×10⁶cells/well, 1.25-2×10⁵ cells/well, or 1×10⁴ cells/well, respectively,for siRNA experiments (densities that allow for some cell death normallyassociated with the electroporation process). Neurons were mixed with 20μL of nucleofection solution (Amaxa) and 600 ng of siRNA (Qiagen orDharmacon). Gene silencing was assayed 3-4 days after plating byquantitative reverse transcriptase polymerase chain reaction (qRT-PCR).Briefly, RNA was isolated from transfected neurons using an RNeasy minikit (Qiagen). Each reaction containing 10 ng of RNA was run intriplicate using the Quantifect SYBR Green RT-PCR kit (Qiagen) withDLK-, JNK1-, JNK2-, or JNK3-specific primers. Primers for thehousekeeping gene, GAPDH, were used as controls. (All primers werepurchased from Qiagen.) Amplification curves were analyzed for relativeRNA concentrations in each sample using ΔCT.

Cortical neurons plated for 3-4 days were treated with 300 nMvincristine, a microtubule destabilizer, for 6-72 hours. NGFwithdrawal-induced apoptosis and degeneration were assayed between 4-36hours after the final treatment. Apoptosis and degeneration were assayedusing both the MTT assay and the lactate dehydrogenase assay. The MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assaymeasures cell viability based on mitochondrial function. MTT was addedto a total of one tenth of the culture volume to each well and incubatedat 37° C. with 5% CO₂ for 90-120 minutes. The MTT-containing culturemedia was aspirated and the cells were resuspended in an equal volume ofsolubilization solution (10% triton X-100, 1 drop HCl, 50 mLisopropanol). Absorbance (570 and 690 nm) was read using aspectrophotometer. Lactate dehydrogenase (LDH) levels were assayed usinga CytoTox non-radioactive LDH assay kit (Promega) to assess the amountof cell death in assays. Fifty μL of media from different treatmentconditions were used according to the manufacturer's instructions. Forboth the MTT and the LDH assays, cell survival/cell death was normalizedto positive controls.

The results are shown in Table 9. As previously observed in theabove-described DLK knockdown, NGF withdrawal experiments, knockdown ofDLK expression was also protective against vincristine-induced corticalneuron death in two different assay systems. This finding indicates thatDLK knockdown may be generally protective against stress-inducedneuronal cell death in a number of different neuron types.

TABLE 9 Effect of DLK Knockdown on Cortical Neuron Survival uponVincristine Challenge as Measured by LDH Assay Condition % cell death(normalized) Control (no treatment) 0 20 hour vincristine treatment 100DLK siRNA (no treatment) 0 DLK siRNA (20 hour vincristine) 11 Condition% Viability (normalized) Control (no treatment) 100 12 hour vincristinetreatment 77 DLK siRNA (12 hr vincristine) 95

C. Effect of DLK Modulation in Sympathetic Neurons

The foregoing analyses demonstrate that DLK inhibition is protectiveagainst toxin-induced or growth factor starvation-induced axondegeneration/apoptosis in cortical neurons and dorsal root ganglionneurons. Further experiments were performed to assess the ability of DLKinhibition to protect sympathetic neurons from the apoptosis normallyinduced by these challenges.

Sympathetic cervical ganglia were dissected from postnatal day 1Sprague-Dawley rats (Charles River Labs) and collected in a dishcontaining Ultraculture media (Lonza) with 5% fetal bovine serum(Invitrogen). Ganglia were washed twice with serum-free Ultraculturemedia, and a final concentration of 0.1% trypsin (Worthington) was addedand incubated with the tissue at 37° C. for 30 minutes. One percentDNase (Roche) solution in PBS was added to the ganglia and incubated atroom temperature for 1-2 minutes. Ganglia were washed with Ultracultureand allowed to settle to the bottom of the tube. All wash media wereremoved and 1 mL plating media containing Ultraculture, 5% rat serum, 1%penicillin-streptomycin, 1% glutamax, 7 μM cytosine arabinoside (Sigma),and 50 ng/mL nerve growth factor (2.5S, Cedarlane Laboratory) was addedto the tube. Ganglia were dissociated by trituration using a pipet(Rainin). The cell slurry was filtered over a 0.45 μm cell strainer(Falcon) and cell number was counted. Sympathetic neurons were plated ata density of 5000-7500 cells/well (96-well dish), 200,000 cells/well(6-well plate), or 100,000 cells/well (8-well chamber slide) and grownfor 4-5 days in 5% CO₂ at 37° C.

Every other day neurons were fed with fresh plating media containing5-10 ng/mL NGF.

NGF withdrawal assays were performed as described in Example 14A.Vincristine treatments, MTT assays, and LDH assays were performed asdescribed in Example 14B.

Lentivirus experiments were performed using two lentivirus vectors:GCMV-MCS-IRES-eGFP and GCMV-MCS-IRES-dsRed. Both vectors are HIV1strains that lack the structural viral genes gag, pol, env, rev, tat,vpr, vif, vpu, and nef. In addition, there is a partial deletion of thepromoter/enhancer sequences within the 3′ LTR that renders the 5′LTR/promoter self-inactivating following integration. The genes providedin trans for both vectors are the structural viral proteins Gag, Pol,Rev, and Tat (via plasmid Delta8.9) and the envelope protein VSV-G.These plasmids are introduced into HEK293-T cells by co-transfection,and transiently express the different viral proteins required togenerate viral particles. The potential for generating wild type orpathogenic lentivirus is extremely low, because it would requiremultiple recombination events amongst three plasmids. In addition, thevirulence factors (vpr, vif, vpu, and nef) have been completely deletedfrom both vectors.

Neurons were infected either immediately at the time of plating or 3-5days after plating by adding virus into cell culture media containing 5μg/mL polybrene. The next day the virus-containing media is removed andreplaced with fresh media. Cells are allowed to express the virus for 48hours before experimentation.

Similar to the protective effect of DLK knockdown on cortical neuronsand DRG, knockdown of DLK protected sympathetic neurons from NGFwithdrawal-induced apoptosis (FIGS. 40A-40B and Table 10) andtoxin-induced apoptosis (FIG. 41 and Table 11).

TABLE 10 DLK Knockdown Protects Sympathetic Neurons from NGF WithdrawalStress (MTT assay) Condition % viability (normalized) Control (with NGF)100 NGF removed 24 hours 46 DLK siRNA control 133 DLK siRNA NGF removed93

TABLE 11 DLK Knockdown Protects Sympathetic Neurons from Toxin-InducedDegeneration (MTT assay) Condition % viability (normalized) Control (notreatment) 100 24 hour vincristine treatment 28 24 hour camptothecintreatment 79 DLK siRNA (24 hour vincristine) 47 DLK siRNA (24 hourcamptothecin) 106Notably, introduction of an excess of kinase-dead DLK was similarlyprotective against NGF withdrawal-induced apoptosis in sympatheticneurons (FIG. 42 and Table 12).

TABLE 12 Dominant Negative DLK was Protective Against NGF Withdrawal-Induced Degeneration in Sympathetic Neurons (MTT assay) Condition %viability (normalized) GFP virus control (with NGF) 77 GFP virus -- NGFremoved 36 DLK virus control (with NGF) 62 DLK virus - NGF removed 22DLK DN virus control (with NGF) 104 DLK DN virus - NGF removed 60

This finding was consistent with the finding in Example 15 thatkinase-dead DLK introduced into 293 cells inhibited JNK phosphorylationin those cells. One non-limiting possibility is that kinase-dead DLK mayprevent normal DLK dimerization and self-phosphorylation. This findingsuggests that inhibition of DLK's JNK-phosphorylating capabilities,either through reduction of DLK expression or through introduction of aDLK variant lacking kinase activity, protects many types of neuronsagainst axon degeneration and apoptosis in response to toxin exposure orgrowth factor withdrawal.

Example 15

Having determined that RNA silencing of DLK and introduction ofkinase-dead DLK were each protective against NGF withdrawal-triggered ortoxin-induced degeneration of cultured neurons, further experiments wereperformed to identify the role of DLK in neuronal degeneration pathways.DLK is a MAP kinase kinase kinase in the multiple lineage kinase (MLK)family. In contrast to other members of the MLK family, the expressionof DLK is restricted to the nervous system (Gallo et al., Nat. Rev. Mol.Cell Biol. 3(9):663-672, 2002; Bisson et al., Cell Cycle 7(7):909-916,2008; Holzman et al., J. Bio. Chem. 269(49):30808-30817, 1994). DLK is amember of a signal transduction pathway activating JNK1-3 and cJun thatresults in apoptosis/inflammation under certain conditions. IncreasedJNK/cJun activity has been linked to a variety of neural disorders,including Parkinson's disease, glaucoma, Alzheimer's disease, ALS,stroke, and Huntington's disease through examination of patient samplesor experiments in animal models of disease (Oo et al., J. Neurochem.72(2):557-564, 1999; Ries et al., J. Neurochem. 107(6):1578-1588, 2008;Vlug et al., Eur. J. Neurosci. 22(8):1881-1894, 2005; Morfini et al.,Nat. Neurosci. 12(7):864-781, 2009; Perrin et al., Exp. Neurol.215(1):191-200, 2009; Levkovitch-Verbin et al., Eye Res. 80(5):663-670,2005; Tezel et al., Brain Res. 996(2):202-212, 2004; Kuan et al., Proc.Natl. Acad. Sci. U.S.A. 100(25):15184-15189, 2003; Yang et al., Nature389(6653):865-870, 1997; Hunot et al., Proc. Natl. Acad. Sci. U.S.A.101(2):665-670, 2004; Thakur et al., J. Neurosci. Res. 85(8):1668-1673,2007). Potential correlation of DLK to neural disorders and theinvolvement of one or more JNK/cJun activation pathways wasinvestigated.

A. Antibodies Specific for Phosphorylated DLK

DLK is a MAP kinase and, like other MAP kinases, it is phosphorylatedwithin the activation loop in its activated state. In order to be ableto distinguish readily between activated and resting (non-activated)DLK, antibodies were generated that are specific for the phosphorylatedform of DLK (“p-DLK”). To do this, specific residues in the active sitewere mutated to alanine, which cannot be phosphorylated. Theseconstructs were then transfected into 293 cells using the same protocolas described in Example 14, part A (above), and tested for theirabilities to phosphorylate the downstream kinase JNK (FIG. 43C). Thisanalysis confirmed that threonine 278 (T278) and serine 281 (S281) wererequired for activity. Antibodies were generated against peptides in theactivation loop of the DLK protein that contained these residues usingstandard in vivo immunization techniques in rabbits. The peptidesequences used for immunization were CKELSDKSpTKMpSFAG (SEQ ID NO: 13)for antibodies 317 and 318, and KMpSFAGTVAWMAKKC (SEQ ID NO: 14) forantibodies 319 and 320, and CGTSKELSDKSpTKM (SEQ ID NO: 15) forantibodies 321 and 322. The procedure was performed at Yenzym, using the“p-site” protocol, which includes purification on columns ofphosphorylated and unphosphorylated peptide to generate selectivity.Polyclonal antibodies from six immunized rabbits were isolated andscreened for binding to DLK and p-DLK.

Six of the obtained polyclonal antibodies were subjected to Western blotand immunohistochemistry analyses to determine their abilities to detectp-DLK, and to distinguish it from non-phosphorylated DLK or otherphosphorylated kinases and phosphorylated MLK3. For cultured cells, themedia was removed and the cells were washed once with ice-cold PBS.Cells were scraped in cold PBS and were transferred to an eppendorf tubeon ice. Cells were quickly pelleted and excess buffer was removed. Thepellets were either snap-frozen on dry ice and stored at −80° C., orwere immediately lysed in fresh lysis buffer (20 mM Tris, 150 mM NaCl,0.1% Triton X-100) containing phosphatase (Sigma P5726 and P2850) andprotease inhibitors (Roche 11836153001). Cells were lysed on a rotatorat 4° C. for 30 minutes and then pelleted at 4° C. Proteinconcentrations were determined using a BCA assay (Pierce).

Protein lysates were mixed with sample buffer (Invitrogen), boiled for 5minutes, and then loaded onto a denaturing 4-12% gradient gel(Invitrogen). Samples were transferred onto nitrocellulose (Invitrogen)and blocked in 5% milk/TBST blocking solution (Tris-bufferedsaline+0.05% Triton X-100) for 1 hour. The blot was incubated with aphospho-DLK antibody at a dilution of 1:1000 in TBST with 5% BSA on ashaking platform overnight at 4° C. Blots were washed three times for 5minutes with TBST, and incubated with a rabbit secondary horseradishperoxidase (HRP)-conjugated antibody (Jackson Labs, 1:5000) for 1-2hours at room temperature. Membranes were washed three times in TBST anddeveloped with Westdura chemiluminescent substrate (Pierce).

293T cells were plated onto coverslips contained in a 12-well culturedish for immunostaining. The cells were then transfected with eithermDLK or mMLK3 as previously described and then fixed with 4% PFA, 30%sucrose 24 hours after transfection. Cells were blocked andpermeabilized with 5% BSA, 0.3% Triton X-100 for an hour, and thenincubated with the primary antibody at 1:500 concentration in 1% BSAovernight. Next the cells were gently rinsed with PBS three times andconjugated with Alexa488 anti-rabbit secondary antibody for 1 hour. Thisstep was followed by three PBS washes, with the final wash containingDAPI (1:5000) to stain nuclei. The coverslip was then carefully liftedout of the culture and mounted onto a slide with the surface with cellsfacing down on the slide, and the slides were visualized usingfluorescence microscopy.

Antibodies 318, 319, 320, 321, and 322 each detected p-DLK andp-DLK/DN-DLK with no (antibodies 318 and 319) or very limited(antibodies 320, 321, and 322) binding to phosphorylated MLK3 (FIG.43A). In contrast, antibody 317 recognized all three proteins.Antibodies 318 and 319 showed the strongest binding to p-DLK, coupledwith minimal p-MLK3 binding, and thus were used for further analysis.

The antibodies were also able to specifically bind to p-DLK in thecontext of a cell, as measured by immunohistochemistry. Both antibodies318 and 319 stained DLK-transfected 293T cells, with significantlyreduced binding observed in MLK3-transfected 293T cells (FIG. 43B). Thisbinding is significantly reduced in 293 cells that have been transfectedwith DLK mutants having a mutation that results in a near complete lackof kinase activity (FIG. 43C). As DLK, like other mixed lineage kinases,is thought to dimerize and autophosphorylate when transfected inheterologous systems, these data further confirm that these antibodiesindeed recognize the phosphorylated form of DLK.

B. Activation of DLK During Vincristine-Induced Degeneration of CorticalNeurons

The antibodies generated as described above were then tested to assesswhether DLK phosphorylation and activity were increased in corticalneurons that had been stressed with vincristine. This would be expected,as knockdown of DLK activity promoted survival. When grown as describedabove and treated with vincristine for 1 hour, levels ofphosphorylated-DLK were elevated as compared to control cultures whenanalyzed by Western blot (same protocol as described above) usingantibodies 318, 319, and 320 (FIG. 43D). This provided furthervalidation that levels of phosphorylated DLK correlate with activity andare increased under conditions of neuronal stress.

C. Expression and Activity of DLK in Neuronal Disease Models

Immunohistochemistry assays were performed as follows. SOD1 transgenicanimals were perfused with 4% paraformaldehyde and the spinal cords werecarefully removed from the vertebral column and post-fixed overnight at4° C. The cords were then equilibrated in 30% sucrose/PBS beforeembedding in OCT for cryosectioning. Coronal sections (20 μm thick) werecut and mounted on cold slides and kept at −80° C. Sections werehydrated in PBS and blocked for endogenous peroxidase activity with H₂O₂(0.3% in PBS) for 10 minutes, followed by rinsing twice with PBST (0.1%Triton X-100). The sections were blocked in 5% BSA, 0.3% Triton X-100 inPBS for 1 hour, and then incubated with primary antibody in p-DLK in 1%BSA, 0.3% Triton X-100 in PBS at 4° C. overnight. Slides were washedthree times in PBST and then labeled with biotinylated secondaryantibody (1:300) in PBS containing 1% BSA, 0.3% Triton X-100 for 1 hour.The slides were rinsed with PBST followed by a 30-minute incubation withavidin DH containing ABS reagent (Vector labs), and finally incubated inperoxidase substrate (0.05% di-amino bendizine in 10 mM Tris, 150 mMNaCl, pH 7.6 to which 30% H₂O₂ was added just before use) in the darkfor 10-15 minutes. The reaction was stopped by rinsing with water, andthe sections were mounted on glass slides and coverslipped. Alzheimer'spatient tissue was purchased from US Biological. Tissue lysate fromhippocampi of Alzheimer's patients and age-matched controls were usedfor this analysis. Analysis was conducted by Western blot using the sameprotocols as described above.

The results in FIG. 44A demonstrate that the levels ofphosphorylated-DLK (p-DLK) were highly elevated in the SOD1 mouse of ALSrelative to wild type mice late in the course of disease. Evidence ofp-DLK was evident in tissue samples as early as 14 weeks (FIG. 44B),which is prior to the onset of ALS-like symptoms in these animals.

Similar results were observed in human Alzheimer's patient samples. Thelevels of p-DLK in cortex samples in Alzheimer's disease patients wereincreased relative to control samples (FIG. 44C). The levels ofphosphorylated JNK and phosphorylated cJun63 levels were also increasedin Alzheimer's disease patients relative to control samples (FIG. 44C).Thus, DLK is activated in a recognized mouse model of ALS, both prior tothe onset of symptoms and at the end stage of disease, and also incortical samples from human Alzheimer's patients.

D. Signaling Contributing to Observed DLK Inhibition Effect

1. Molecular Effect of DLK Silencing

As evidenced above in Example 14, DLK silencing protects neurons fromdegradation in response to toxin exposure or growth factor withdrawal,and DLK kinase activity is involved directly or indirectly in JNKphosphorylation. Experiments were performed to assess JNK and cJunexpression and activity under NGF withdrawal stress and toxin-dependentstress conditions. The experimental protocols

used to decrease the expression of DLK (siRNA) and for immunoblottingwere the same as those described above.

Silencing of DLK did not change baseline JNK protein levels or JNKactivity in DRGs or cortical neurons (FIG. 45). DLK knockdown howeverdecreased p-cJun levels after either NGF withdrawal orvincristine-dependent stress in cortical neurons, DRGS, and sympatheticganglia neurons (FIG. 45). DLK knockdown also decreased p-JNK levelsafter either NGF withdrawal or vincristine-dependent stress in corticalneurons and DRG (FIG. 45). It should be noted that the p-JNK antibodyrecognizes the phosphorylated forms of each of JNK1, JNK2, and JNK3.JNK1 phosphorylation does not correlate with stress induction orphosphorylation of cJun. DLK knockdown silences stress-inducedphosphorylation of JNK2 and JNK3. The background signal observed in thecontrol samples in these data is likely due to baseline phosphorylatedJNK1. The results show that DLK silencing or other inhibition does notcause degradation of JNK or cJun, but does result in a lesser degree ofstress-induced phosphorylation (and thus lack of activation) of thosemolecules.

If DLK inhibition protects neurons from degeneration through limitingthe phosphorylation of JNK2 and JNK3, then inhibitors of JNK2 or JNK3should also protect neurons from cell death and axon degeneration. Theability of direct JNK inhibition to protect neurons against NGFwithdrawal-dependent degeneration was also assessed. DRG explants weretreated with anti-NGF and either pan-JNK inhibitor JNKV or JNKVIII,prior to assessment in the NGF withdrawal assay described above. Bothinhibitors were able to protect DRG explants from NGF-dependentdegeneration (FIG. 46): JNKV (Calbiochem) was most protective at aconcentration of 5 μM, and JNKVIII (Calbiochem) at a concentration of 4μM.

To identify which JNK(s) were involved in the observed protectiveeffect, further knockdown experiments were performed using the samemethodology as described above. Silencing RNAs specific for JNK1 alonewere not able to replicate the effects observed with the pan-JNKinhibitors (FIG. 47). Inhibitory RNAs (siRNAs) for JNK2 or JNK3 alonewere not able to protect DRG against axon degeneration upon NGFwithdrawal (FIG. 47). The most effective protection was observed in thepresence of silencing of both JNK2 and JNK3, though even this protectionwas not the same extent as siDLK alone (FIG. 47).

JNK1 is a constitutively active molecule, known to be responsible foraxon maintenance and synapse maturation, whereas JNK2 and JNK3 are knownto be stress-induced and involved in cJun activation and apoptosis/axondegeneration (Coffey et al., J. Neurosci. 22(11):4335-4345, 2002; Coffeyet al., J. Neurosci. 20(20):7602-7613, 2000). The fact that DLKinhibition results in a larger neuroprotective effect than JNK1/2/3inhibition strongly suggests that DLK is inhibitory to one or moremechanisms of neuron growth and/or survival in addition to its role inthe JNK/cJUN pathway.

E. Trophic Effects of DLK

A previous study has suggested that a dominant negative kinase-deadmutant form of DLK (K152A) causes trophic effects in dopaminergicneurons of the substantia nigra pars compacta, whereas a dominantnegative form of DLK containing only the leucine zipper domain does notcause such effects (Chen et al., J. Neurosci. 28(3):672-680, 2008).Notably, however, trophism in this study was defined by an increasednumber of DLK (K152A) infected neurons expressing a particular markerrather than any functional or morphological differences between infectedand non-infected neurons. The impact of DLK silencing was accordinglyinvestigated in cortical neurons and DRGs. The protocols used for thesestudies were the same as those described above.

DLK knockdown was shown to have pronounced trophic effects in bothcortical neurons and sympathetic neurons (observed increase in number ofcells) (FIGS. 48A and 48B). Specifically, DRG, cortical, and sympatheticneurons showed increased growth/survival in culture when treated withsiRNA directed against DLK (observed increase in growth) (Table 13).

TABLE 13 Increased Growth of Multiple Types of Neurons in Culture afterDLK Knockdown (MTT assay) Condition % viability (normalized) Corticalneurons control 100 Cortical neurons + DLK siRNA 137 Sympathetic neuronscontrol 100 Sympathetic neurons + DLK siRNA 139 DRG neurons 100 DRGneurons + DLK siRNA 147

To further examine the role of DLK in neuronal growth, the levels ofMAP2, were measured, a microtubule associated protein that isspecifically localized to dendrites. MAP2 expression in cultured neuronsis reflective of neuronal maturation and expression, and is oftenincreased under pro-growth conditions. Inhibition of DLK activity usingeither a dominant-negative leucine zipper form of DLK or a dominantnegative kinase-dead form of DLK in neurons resulted in induction ofMAP2 protein production. The induction of MAP2 expression indicates thatneuronal growth and maturation are taking place, and further confirmsthat DLK knockdown indeed has a functional trophic effect on neurons.

Example 16

The assays described herein identified several inhibitors of G-proteinsand G-protein coupled receptors (GPCRs) that decrease degeneration inneurons. Experiments were performed in DRGs to verify the role ofG-proteins and GPCRs in degeneration pathways. The experimentalprotocols used in these experiments are the same as those described inExample 14. Pertussis toxin and SCH 202676 were purchased from TocrisBioscience.

The data demonstrate that SCH 202676, a sulphydryl-reactive compoundthat inhibits agonist and antagonist binding to G protein-coupledreceptors, decreases NGF withdrawal-induced degeneration in DRGs (FIGS.49 and 50). Pertussis toxin, another inhibitor of G-protein signaling,also decreases degeneration in neurons following NGF withdrawal (FIG.51). These data indicate that G-proteins and G-protein coupled receptorsplay a role in neuron degeneration.

Example 17

Additional cellular targets, identified in the assays described herein,and which play a role in degeneration pathways, are members of thebeta-catenin signaling pathway and its downstream targets (e.g.,transcription factor 4, TCF4). Experiments were performed in hippocampalneurons to verify the role of beta-catenin and TCF4 in neurondegeneration. The experimental protocols used in these experiments arethe same as those described in Example 14.

The data show that expression of a constitutively active GSK3, which canresult in loss of beta-catenin, mediates a decrease in neuron viability(FIG. 52; top right panel). The expression of a dominant negative formof TCF4 also mediates a decrease in neuron viability (FIG. 52, bottomright panel). These data indicate that the beta-catenin signalingpathway is important for the maintenance of neuron viability. Inhibitorsthat target the beta-catenin signaling pathway can be used to promoteneuron degeneration (e.g., inhibitors of beta-catenin expression andinhibitors of TCF4 activity.) Conversely, activators or beta-catenin andTCF4 signaling may prevent axon degeneration and neuronal cell death.

Other Embodiments

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. Use of singular forms herein, such as “a” and “the,” does notexclude indication of the corresponding plural form, unless the contextindicates to the contrary. Although the invention has been described insome detail by way of illustration and example for purposes of clarityof understanding, it will be readily apparent to those of ordinary skillin the art in light of the teachings of the invention that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims.

What is claimed is:
 1. A pharmaceutical composition or kit comprisingone or more agents that can be used to inhibit degeneration of a neuronor portion thereof, wherein the one or more agents are one or more shortinterfering RNA (siRNA) molecules that target dual leucinezipper-bearing kinase (DLK) and comprise a nucleic acid sequenceselected from the group consisting of GCACTGAATTGGACAACTCTT (SEQ ID NO:1), GAGTTGTCCAATTCAGTGCTT (SEQ ID NO: 2), GGACATCGCCTCCGCTGATTT (SEQ IDNO: 3), ATCAGCGGAGGCGATGTCCTT (SEQ ID NO: 4), GCAAGACCCGTCACCGAAATT (SEQID NO: 5), TTTCGGTGACGGGTCTTGCTT (SEQ ID NO: 6), GCGGTGTCCTGGTCTACTATT(SEQ ID NO: 7), and TAGTAGACCAGGACACCGCTT (SEQ ID NO: 8).
 2. Thepharmaceutical composition or kit of claim 1, further comprising one ormore pharmaceutically acceptable excipients or instructions for use ofthe composition or kit in a method for inhibiting degeneration of aneuron or portion thereof.